Effects of perinatal stress and maternal traumatic stress on the cortisol regulation of preterm infants.

Preterm infants experience intense stress during the perinatal period because they endure painful and intense medical procedures. Repeated activation of the hypothalamic-pituitary-adrenal (HPA) axis during this period may have long-term effects on subsequent cortisol regulation. A premature delivery may also be intensely stressful for the parents, and they may develop symptoms of posttraumatic stress disorder (PTSD). Usable saliva samples were collected (4 times per day over 2 days, in the morning at awakening, at midday, in the afternoon, and in the evening before going to bed) to assess the diurnal cortisol regulation from 46 preterm infants when the infants were 12 months of corrected age (∼ 14 months after birth). Mothers reported their level of PTSD symptoms. The results showed an interaction between perinatal stress and maternal traumatic stress on the diurnal cortisol slope of preterm infants (R(2) = .32). This suggests that the HPA axis of preterm infants exposed to high perinatal stress may be more sensitive to subsequent environmental stress.

A Dose-Response Strategy Reveals Differences between Normal-Weight and Obese Men in Their Metabolic and Inflammatory Responses to a High-Fat Meal.

A dose-response strategy may not only allow investigation of the impact of foods and nutrients on human health but may also reveal differences in the response of individuals to food ingestion based on their metabolic health status. In a randomized crossover study, we challenged 19 normal-weight (BMI: 20-25 kg/m(2)) and 18 obese (BMI: >30 kg/m(2)) men with 500, 1000, and 1500 kcal of a high-fat (HF) meal (60.5% energy from fat). Blood was taken at baseline and up to 6 h postprandially and analyzed for a range of metabolic, inflammatory, and hormonal variables, including plasma glucose, lipids, and C-reactive protein and serum insulin, glucagon-like peptide-1, interleukin-6 (IL-6), and endotoxin. Insulin was the only variable that could differentiate the postprandial response of normal-weight and obese participants at each of the 3 caloric doses. A significant response of the inflammatory marker IL-6 was only observed in the obese group after ingestion of the HF meal containing 1500 kcal [net incremental AUC (iAUC) = 22.9 ± 6.8 pg/mL × 6 h, P = 0.002]. Furthermore, the net iAUC for triglycerides significantly increased from the 1000 to the 1500 kcal meal in the obese group (5.0 ± 0.5 mmol/L × 6 h vs. 6.0 ± 0.5 mmol/L × 6 h; P = 0.015) but not in the normal-weight group (4.3 ± 0.5 mmol/L × 6 h vs. 4.8 ± 0.5 mmol/L × 6 h; P = 0.31). We propose that caloric dose-response studies may contribute to a better understanding of the metabolic impact of food on the human organism. This study was registered at clinicaltrials.gov as NCT01446068.

Phosphatidyl inositol 3-kinase signaling in hypothalamic proopiomelanocortin neurons contributes to the regulation of glucose homeostasis.

Recent studies demonstrated a role for hypothalamic insulin and leptin action in the regulation of glucose homeostasis. This regulation involves proopiomelanocortin (POMC) neurons because suppression of phosphatidyl inositol 3-kinase (PI3K) signaling in these neurons blunts the acute effects of insulin and leptin on POMC neuronal activity. In the current study, we investigated whether disruption of PI3K signaling in POMC neurons alters normal glucose homeostasis using mouse models designed to both increase and decrease PI3K-mediated signaling in these neurons. We found that deleting p85alpha alone induced resistance to diet-induced obesity. In contrast, deletion of the p110alpha catalytic subunit of PI3K led to increased weight gain and adipose tissue along with reduced energy expenditure. Independent of these effects, increased PI3K activity in POMC neurons improved insulin sensitivity, whereas decreased PI3K signaling resulted in impaired glucose regulation. These studies show that activity of the PI3K pathway in POMC neurons is involved in not only normal energy regulation but also glucose homeostasis.

Tumor necrosis factor-alpha and alphaB-crystallin up-regulation during antibody-mediated demyelination in vitro: a putative protective mechanism in oligodendrocytes.

By using an in vitro model of antibody-mediated demyelination, we investigated the relationship between tumor necrosis factor-alpha (TNF-alpha) and heat shock protein (HSP) induction with respect to oligodendrocyte survival. Differentiated aggregate cultures of rat telencephalon were subjected to demyelination by exposure to antibodies against myelin oligodendrocyte glycoprotein (MOG) and complement. Cultures were analyzed 48 hr after exposure. Myelin basic protein (MBP) expression was greatly decreased, but no evidence was found for either necrosis or apoptosis. TNF-alpha was significantly up-regulated. It was localized predominantly in neurons and to a lesser extent in astrocytes and oligodendrocytes, and it was not detectable in microglial cells. Among the different HSPs examined, HSP32 and alphaB-crystallin were up-regulated; they may confer protection from oxidative stress and from apoptotic death, respectively. These results suggest that TNF-alpha, often regarded as a promoter of oligodendroglial death, could alternatively mediate a protective pathway through alphaB-crystallin up-regulation.

Plasma membrane H+-ATPase regulation is required for auxin gradient formation preceding phototropic growth.

Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H(+)-ATPases that are required to control apoplastic pH. Our results show that H(+)-ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H(+)-ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH.

ON THE ROLE OF PERIOD-2 IN THE CIRCADIAN AND HOMEOSTATIC REGULATION OF SLEEP

Humans spend one third of their life sleeping, then we could raise the basic question: Why do we sleep? Despite the fact that we still don't fully understand its function, we made much progress in understanding at different levels how sleep is regulated. One model suggests that sleep is regulated by two processes: a homeostatic process that tracks the need for sleep and by a circadian rhythm that determines the preferred time-of-day sleep occurs. At the molecular level circadian rhythms are a property of interlocking transcriptional regula-tors referred to as clock genes. The heterodimeric transcription factors BMAL1::CLOCK/NPAS2 drive the transcription of many target genes including the clock genes Cryptochome1 (Cry1), Cry2, Period1 (Per1), and Per2. The encoded CRY/PER proteins are transcriptional inhibitors of BMAL1::CLOCK/NPAS2 thereby providing negative feedback to their own transcription. These genes seem, however, also involved in sleep homeostasis because the brain expression of clock genes, es-pecially that of Per2, increase as a function of time-spent-awake and because mice lacking clock genes display altered sleep homeostasis. The aim of first part of my doctoral work has been to advance our understanding the link that exists between sleep homeostasis and circadian rhythms investigating a possible mechanism by which sleep deprivation could alter clock gene expression by quantifying DNA-binding of the core-clock genes BMAL1, CLOCK and NPAS2 to their target chromatin loci including the E-box enhancers of the Per2 promoter. We made use of chromatin immunoprecipitation (ChIP) and quantitative poly-merase chain reaction (qPCR) to show that DNA-binding of CLOCK and BMAL1 to their target genes changes as a function of time-of-day in both liver and cerebral cortex. We then performed a 6h sleep deprivation (SD) and observed a significant decrease in DNA-binding of CLOCK and BMAL1 to Dbp. This is consistent with a decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was similarly decreased following SD. However, SD has been previously shown to in-crease Per2 expression in the cortex which seems paradoxical. Our results demonstrate that sleep-wake history can affect the molecular clock machinery directly at the level of the chromatin thereby altering the cortical expression of Dbp and Per2, and likely other targets. However, the precise dy-namic relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive. The second aim of my doctoral work has been to perform an in depth characterization of cir-cadian rhythmicity, sleep architecture, analyze the response to SD in full null-Per2 knock-out (Per2-/-) mice, and Per1-/- mice, as well as their double knock-out offspring (Per1,2-/-) and littermate wildtype (Wt) mice. The techniques used include locomotor activity recording by passive infrared (PIR) sen-sors, EEG/EMG surgery, recording, and analysis, and cerebral cortex extraction and quantification of mRNA levels by qPCR. Under standard LD12:12 conditions, we found that wakefulness onset, as well as the time courses of clock gene expression in the brain and corticosterone plasma levels were ad-vanced by about 2h in Per2-/- mice compared to Wt mice. When released under constant dark condi-tions almost all Per2-/- mice (97%) became arrhythmic immediately. From these observations, we conclude that while Per2-/- mice seem to be able to anticipate dark onset, this does not result from a self-sustained circadian clock. Our results suggest instead that the earlier onset of activity results from a labile, not-self sustained 22h rhythm linked to light onset suggesting the existence of a light-driven rhythm. Analyses of sleep under LD12:12 conditions revealed that in both Per2-/- and Per1,2-/- mice the same sleep phenotypes are observed compared to Wt mice: increased NREM sleep frag-mentation and inability to adequately compensate the loss of NREM sleep. That suggests a possible role of PER2 in sleep consolidation and recovery.

Glucose sensing and the pathogenesis of obesity and type 2 diabetes.

The control of body weight and of blood glucose concentrations depends on the exquisite coordination of the function of several organs and tissues, in particular the liver, muscle and fat. These organs and tissues have major roles in the use and storage of nutrients in the form of glycogen or triglycerides and in the release of glucose or free fatty acids into the blood, in periods of metabolic needs. These mechanisms are tightly regulated by hormonal and nervous signals, which are generated by specialized cells that detect variations in blood glucose or lipid concentrations. The hormones insulin and glucagon not only regulate glycemic levels through their action on these organs and the sympathetic and parasympathetic branches of the autonomic nervous system, which are activated by glucose or lipid sensors, but also modulate pancreatic hormone secretion and liver, muscle and fat glucose and lipid metabolism. Other signaling molecules, such as the adipocyte hormones leptin and adiponectin, have circulating plasma concentrations that reflect the level of fat stored in adipocytes. These signals are integrated at the level of the hypothalamus by the melanocortin pathway, which produces orexigenic and anorexigenic neuropeptides to control feeding behavior, energy expenditure and glucose homeostasis. Work from several laboratories, including ours, has explored the physiological role of glucose as a signal that regulates these homeostatic processes and has tested the hypothesis that the mechanism of glucose sensing that controls insulin secretion by the pancreatic beta-cells is also used by other cell types. I discuss here evidence for these mechanisms, how they integrate signals from other nutrients such as lipids and how their deregulation may initiate metabolic diseases.

Re-thinking cell cycle regulators: the cross-talk with metabolism.

Analysis of genetically engineered mice deficient in cell cycle regulators, including E2F1, cdk4, and pRB, showed that the major phenotypes are metabolic perturbations. These key cell cycle regulators contribute to lipid synthesis, glucose production, insulin secretion, and glycolytic metabolism. It has been shown that deregulation of these pathways can lead to metabolic perturbations and related metabolic diseases, such as obesity and type II diabetes. The cyclin-cdk-Rb-E2F1 pathway regulates adipogenesis in addition to its well-described roles in cell cycle regulation and cancer. It was also shown that E2F1 directly participates in the regulation of pancreatic growth and function. Similarly, cyclin D3, cdk4, and cdk9 are also adipogenic factors with strong effects on whole organism metabolism. These examples support the emerging notion that cell cycle regulatory proteins also modulate metabolic processes. These cell cycle regulators are activated by insulin and glucose, even in non-proliferating cells. Most importantly, these cell cycle regulators trigger the adaptive metabolic switch that normal and cancer cells require in order to proliferate. These changes include increased lipid synthesis, decreased oxidative metabolism, and increased glycolytic metabolism. In summary, these factors are essential regulators of anabolic biosynthetic processes, blocking at the same time oxidative and catabolic pathways, which is reminiscent of cancer cell metabolism.

Spatially and genetically distinct control of seed germination by phytochromes A and B.

Phytochromes phyB and phyA mediate a remarkable developmental switch whereby, early upon seed imbibition, canopy light prevents phyB-dependent germination, whereas later on, it stimulates phyA-dependent germination. Using a seed coat bedding assay where the growth of dissected embryos is monitored under the influence of dissected endosperm, allowing combinatorial use of mutant embryos and endosperm, we show that canopy light specifically inactivates phyB activity in the endosperm to override phyA-dependent signaling in the embryo. This interference involves abscisic acid (ABA) release from the endosperm and distinct spatial activities of phytochrome signaling components. Under the canopy, endospermic ABA opposes phyA signaling through the transcription factor (TF) ABI5, which shares with the TF PIF1 several target genes that negatively regulate germination in the embryo. ABI5 enhances the expression of phytochrome signaling genes PIF1, SOMNUS, GAI, and RGA, but also of ABA and gibberellic acid (GA) metabolic genes. Over time, weaker ABA-dependent responses eventually enable phyA-dependent germination, a distinct type of germination driven solely by embryonic growth.

Regulation of the Activity of the Dual-Function DnaA Protein in Caulobacter crescentus.

DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A)]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA). We found that the expression of the DnaA(R357A) mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A) protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A) could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

Regulation of lymphocyte proliferation and death by FLIP.

Lymphocyte homeostasis is a balance between lymphocyte proliferation and lymphocyte death. Tight control of apoptosis is essential for immune function, because its altered regulation can result in cancer and autoimmunity. Signals from members of the tumour-necrosis-factor receptor (TNF-R) family, such as Fas and TNF-R1, activate the caspase cascade and result in lymphocyte death by apoptosis. Anti-apoptotic proteins, such as FLIP (also known as FLICE/caspase-8 inhibitory protein) have recently been identified. FLIP expression is tightly regulated in T cells and might be involved in the control of both T-cell activation and death. Abnormal expression of FLIP might have a role not only in autoimmune diseases, but also in tumour development and cardiovascular disorders.

Polymers of 3-hydroxyacids in plants

Polyhydroxyalkanoates (PHA) are polyesters of bacterial origin that have properties of biodegradable plastics and elastomers. Synthesis of PHA in crop plants would allow the large-scale production and use of these biodegradable and renewable polymers as substitutes for petroleum-derived plastics. Synthesis of a diversity of PHAs in plants, such as Arabidopsis thaliana, rapeseed, corn and cotton, has been demonstrated through the genetic engineering of metabolic pathways in the cytoplasm, plastid and peroxisome. PHA can also be used as a novel tool to study various aspects of plant metabolism, such as the regulation of carbon flux to the fatty acid biosynthetic and degradation pathways.

Role of atrial natriuretic peptides and neuropeptide Y in blood pressure regulation.

Atrial natriuretic peptides (ANP) are released into the circulation in response to enhanced atrial stretching. These peptides not only have diuretic and natriuretic properties, but also exert a relaxing effect on the vasculature. Moreover, they antagonize the contractions induced by norepinephrine and angiotensin II. Neuropeptide Y (NPY) is also a vasoactive peptide. It is widely distributed throughout the central and peripheral nervous systems. NPY is coreleased with norepinephrine by perivascular nerve endings. At high concentrations, this peptide has a direct vasoconstrictor effect. In addition, it enhances the vascular effect of various agonists, including norepinephrine and angiotensin II. Both ANP and NPY have an inhibitory effect on renin secretion. This effect may have important implications for the role of these peptides in cardiovascular regulation.

Neonatal steroids induce a down-regulation of tenascin-C and elastin and cause a deceleration of the first phase and an acceleration of the second phase of lung alveolarization.

Pre- and postnatal corticosteroids are often used in perinatal medicine to improve pulmonary function in preterm infants. To mimic this clinical situation, newborn rats were treated systemically with dexamethasone (Dex), 0.1-0.01 mg/kg/day on days P1-P4. We hypothesized that postnatal Dex may have an impact on alveolarization by interfering with extracellular matrix proteins and cellular differentiation. Morphological alterations were observed on 3D images obtained by high-resolution synchrotron radiation X-ray tomographic microscopy. Alveolarization was quantified stereologically by estimating the formation of new septa between days P4 and P60. The parenchymal expression of tenascin-C (TNC), smooth muscle actin (SMA), and elastin was measured by immunofluorescence and gene expression for TNC by qRT-PCR. After Dex treatment, the first phase of alveolarization was significantly delayed between days P6 and P10, whereas the second phase was accelerated. Elastin and SMA expressions were delayed by Dex treatment, whereas TNC expression was delayed and prolonged. A short course of neonatal steroids impairs the first phase of alveolarization, most likely by altering the TNC and elastin expression. Due to an overshooting catch-up during the second phase of alveolarization, the differences disappear when the animals reach adulthood.