Intratumoral heterogeneity in glioblastoma: don't forget the peritumoral brain zone

International audience

Observed Social Problem Solving and Friendship Quality in Children with a Traumatic Brain Injury

Children who have experienced a traumatic brain injury (TBI) are at risk for a variety of maladaptive cognitive, behavioral and social outcomes (Yeates et al., 2007). Research involving the social problem solving (SPS) abilities of children with TBI indicates a preference for lower level strategies when compared to children who have experienced an orthopedic injury (OI; Hanten et al., 2008, 2011). Research on SPS in non-injured populations has highlighted the significance of the identity of the social partner (Rubin et al., 2006). Within the pediatric TBI literature few studies have utilized friends as the social partner in SPS contexts, and fewer have used in-vivo SPS assessments. The current study aimed to build on existing research of SPS in children with TBI by utilizing an observational coding scheme to capture in-vivo problem solving behaviors between children with TBI and a best friend. The current study included children with TBI (n = 41), children with OI (n = 43), and a non-injured typically developing group (n = 41). All participants were observed completing a task with a friend and completed a measure of friendship quality. SPS was assessed using an observational coding scheme that captured SPS goals, strategies, and outcomes. It was expected children with TBI would produce fewer successes, fewer direct strategies, and more avoidant strategies. ANOVAs tested for group differences in SPS successes, direct strategies and avoidant strategies. Analyses were run to see if positive or negative friendship quality moderated the relation between group type and SPS behaviors. Group differences were found between the TBI and non-injured group in the SPS direct strategy of commands. No group differences were found for other SPS outcome variables of interest. Moderation analyses partially supported study hypotheses regarding the effect of friendship quality as a moderator variable. Additional analyses examined SPS goal-strategy sequencing and grouped SPS goals into high cost and low cost categories. Results showed a trend supporting the hypothesis that children with TBI had fewer SPS successes, especially with high cost goals, compared to the other two groups. Findings were discussed highlighting the moderation results involving children with severe TBI.

Vocal differentiation parallels development of auditory saccular sensitivity in a highly soniferous fish

Vocal differentiation is widely documented in birds and mammals but has been poorly investigated in other vertebrates, including fish, which represent the oldest extant vertebrate group. Neural circuitry controlling vocal behaviour is thought to have evolved from conserved brain areas that originated in fish, making this taxon key to understanding the evolution and development of the vertebrate vocal-auditory systems. This study examines ontogenetic changes in the vocal repertoire and whether vocal differentiation parallels auditory development in the Lusitanian toadfish Halobatrachus didactylus (Batrachoididae). This species exhibits a complex acoustic repertoire and is vocally active during early development. Vocalisations were recorded during social interactions for four size groups (fry: <2 cm; small juveniles: 2-4 cm; large juveniles: 5-7 cm; adults >25 cm, standard length). Auditory sensitivity of juveniles and adults was determined based on evoked potentials recorded from the inner ear saccule in response to pure tones of 75-945 Hz. We show an ontogenetic increment in the vocal repertoire from simple broadband-pulsed 'grunts' that later differentiate into four distinct vocalisations, including low-frequency amplitude-modulated 'boatwhistles'. Whereas fry emitted mostly single grunts, large juveniles exhibited vocalisations similar to the adult vocal repertoire. Saccular sensitivity revealed a three-fold enhancement at most frequencies tested from small to large juveniles; however, large juveniles were similar in sensitivity to adults. We provide the first clear evidence of ontogenetic vocal differentiation in fish, as previously described for higher vertebrates. Our results suggest a parallel development between the vocal motor pathway and the peripheral auditory system for acoustic social communication in fish.

Modulation of GABA and glutamate release by brain-derived neurotrophic factor : role of adenosine A2A receptors

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Fiber Pathways for Language in the Developing Brain: A Diffusion Tensor Imaging (DTI) Study

The present study characterized two fiber pathways important for language, the superior longitudinal fasciculus/arcuate fasciculus (SLF/AF) and the frontal aslant tract (FAT), and related these tracts to speech, language, and literacy skill in children five to eight years old. We used Diffusion Tensor Imaging (DTI) to characterize the fiber pathways and administered several language assessments. The FAT was identified for the first time in children. Results showed no age-related change in integrity of the FAT, but did show age-related change in the left (but not right) SLF/AF. Moreover, only the integrity of the right FAT was related to phonology but not audiovisual speech perception, articulation, language, or literacy. Both the left and right SLF/AF related to language measures, specifically receptive and expressive language, and language content. These findings are important for understanding the neurobiology of language in the developing brain, and can be incorporated within contemporary dorsal-ventral-motor models for language.

SOLUTIONS: Assistive Technology for People with Brain Injury, February 22, 2016

The Brain A project of the Iowa Department of Public Health and the Iowa Advisory Council on Brain Injuries, produced with assistance from the Iowa Program for Assistive Technology University of Iowa Center for Disabilities and Development and Easter Seals This booklet was supported in part by the Health Resources and Services Administration (HRSA) of the U.S. Department of Health and Human Services (HHS) under grant number H21MC26929 titled: Traumatic Brain Injury Implementation. This information or content and conclusions are those of the authors/s and should not be construed as the official position or policy of, nor should any endorsements be inferred by, HRSA, HHS, or the U.S. Government.

Alterations of the epigenome in brain cancer: loss of 5-hydroxymethylcytosine

Abstract : 5-Methylcytosine is an epigenetic mark, which can be oxidized to 5-hydroxymethylcytosine (5hmC) in DNA by ten-eleven translocation (TET) oxygenases. It is an initial step in the demethylation of 5mC. Levels of 5hmC is relatively high in the brain compared to other organs, but these levels are known to be significantly reduced during the development of a brain tumor, especially in glioblastoma multiforme (GBM). However, no known mechanisms may fully explain this abnormality. The objectives of my project were to (1) understand the implications of the demethylation pathway mediated by TET, and (2) gain a deeper insight in the epigenetic make-up of brain tumors. (1) U87 cells were incubated with 5mC, 5hmC, 5-formylcytosine (5fC) or co-incubated of 5hmC with 3,4,5,6-tetrahydro-2’-deoxyuridine (dTHU) over a timeline of 0, 24, 48 and 96 hours. (2) 130 brain tumors (GBM= 79; grade II/III= 51) were obtained directly from surgery and immediately suspended in DNA extraction buffer. Both cell samples and tumor tissues underwent DNA extraction and DNA digestion protocols. The percent per cytosine (%/C) was obtained by quantification of 5mC, 5hmC, 5fC, 5-hydroxymethyluracil (5hmU) and 5formyluracil (5fU) using LC-MS/MS. (1) Cellular incubations showed that it is possible to increase levels of 5hmC in DNA, but also a slight increase in 5mC levels throughout the experiment. 5HmC levels dramatically increased by 1.9-fold after 96h. On the other hand, no increase was observed in 5fC levels. Both 5hmC and 5fC incubations were accompanied by high increases in 5hmU and 5fU levels respectively. The addition of dTHU to the 5hmC incubation decreased 5hmU incorporation by 65%. (2) The average levels of 5mC, 5hmC and 5fC, in brain tumors, were 4.0, 0.15 and 0.021 %/C respectively. 5HmU and 5fU levels were present at comparable levels of 5hmC and 5fC. Levels of 5hmC, 5hmU and 5fU were significantly lower in the DNA of GBM specimens. There was a strong correlation between 5mC with 5hmC and 5fC in GBM, but this was absent in low grade tumors. The presence of 5hmU and 5fU in brain tumor and the increase in their levels during cell incubations indicate a deamination activity in these cancerous cells, which may impinge on the cellular levels of 5hmC, in particular. Furthermore, upon the incubations with 5hmC, downstream levels of 5fC did not increase suggesting a TET malfunction. TET activity is maintained in GBMs, but impaired in low grade tumors due to isocitrate dehydrogenase-1 (IDH1) mutations. Therefore, in brain tumors, a strong deamination activity and TET impairment may lead to epigenetic reduction of 5hmC.

Development of an innovative strategy to enhance the efficacy of gene therapy for CDKL5 deficiency disorder

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental disease caused by mutations in the CDKL5 gene, characterized by early-onset epileptic seizures, intellectual disability, motor and visual impairment and respiratory dysregulation. Although pharmacological treatments are used to control seizures, there is currently no cure to ameliorate symptoms for CDD. Albeit delivery of a wild-type copy of the mutated gene to cells represents the most curative approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for brain gene therapy. The major one regards the low efficiency of gene delivery to the CNS by viral vectors. We used a secretable Igk-TATk-CDKL5 protein to enhance the efficiency of a gene therapy for CDD. In view of the properties of the Igk-chain leader sequence, the TATk-CDKL5 protein produced by infected cells is secreted via constitutive secretory pathways. Importantly, due to the transduction property of the TATk peptide, the secreted CDKL5 protein is internalized by cells. We compared the effects of a CDKL5 gene therapy with an IgK-TATk-CDKL5 gene therapy in a Cdkl5 KO mouse model to validate whether the Igk-TATk-CDKL5 approach significantly improve the therapeutic efficacy. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to a higher CDKL5 protein replacement and Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with Cdkl5 KO mice treated with the AAVPHP.B_CDKL5 vector.

Development of advanced analytical methodologies for Alzheimer’s disease drug discovery

Advanced analytical methodologies were developed to characterize new potential active MTDLs on isolated targets involved in the first stages of Alzheimer’s disease (AD). In addition, the methods investigated drug-protein bindings and evaluated protein-protein interactions involved in the neurodegeneration. A high-throughput luminescent assay allowed the study of the first in class GSK-3β/ HDAC dual inhibitors towards the enzyme GSK-3β. The method was able to identify an innovative disease-modifying agent with an activity in the micromolar range both on GSK-3β, HDAC1 and HDAC6. Then, the same assay reliably and quickly selected true positive hit compounds among natural Amaryllidaceae alkaloids tested against GSK-3β. Hence, given the central role of the amyloid pathway in the multifactorial nature of AD, a multi-methodological approach based on mass spectrometry (MS), circular dichroism spectroscopy (CD) and ThT assay was applied to characterize the potential interaction of CO releasing molecules (CORMs) with Aβ1-42 peptide. The comprehensive method provided reliable information on the different steps of the fibrillation process and regarding CORMs mechanism of action. Therefore, the optimal CORM-3/Aβ1−42 ratio in terms of inhibitory effect was identified by mass spectrometry. CD analysis confirmed the stabilizing effect of CORM-3 on the Aβ1−42 peptide soluble form and the ThT Fluorescent Analysis ensured that the entire fibrillation process was delayed. Then the amyloid aggregation process was studied in view of a possible correlation with AD lipid brain alterations. Therefore, SH-SY5Y cells were treated with increasing concentration of Aß1-42 at different times and the samples were analysed by a RP-UHPLC system coupled with a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode. Each lipid class profiling in SH-SY5Y cells treated with Aß1-42 was compared to the one obtained from the untreated. The approach underlined some peculiar lipid alterations, suitable as biomarkers, that might be correlated to Aß1-42 different aggregation species.

Noises: a Nuisance and a Resource. Development of New Decomposition Methods of Noisy Data

Noise is constant presence in measurements. Its origin is related to the microscopic properties of matter. Since the seminal work of Brown in 1828, the study of stochastic processes has gained an increasing interest with the development of new mathematical and analytical tools. In the last decades, the central role that noise plays in chemical and physiological processes has become recognized. The dual role of noise as nuisance/resource pushes towards the development of new decomposition techniques that divide a signal into its deterministic and stochastic components. In this thesis I show how methods based on Singular Spectrum Analysis have the right properties to fulfil the previously mentioned requirement. During my work I applied SSA to different signals of interest in chemistry: I developed a novel iterative procedure for the denoising of powder X-ray diffractograms; I “denoised” bi-dimensional images from experiments of electrochemiluminescence imaging of micro-beads obtaining new insight on ECL mechanism. I also used Principal Component Analysis to investigate the relationship between brain electrophysiological signals and voice emission.

Development of functional MRI protocols in the study of neurodegenerative diseases: MRI study in patients with REM sleep behavior disorder, idiopathic Parkinson's disease and multisystem atrophy

In the central nervous system, iron in several proteins is involved in many important processes: oxygen transportation, oxidative phosphorylation, mitochondrial respiration, myelin production, the synthesis and metabolism of neurotransmitters. Abnormal iron homoeostasis can induce cellular damage through hydroxyl radical production, which can cause the oxidation, modification of lipids, proteins, carbohydrates, and DNA, lead to neurotoxicity. Moreover increased levels of iron are harmful and iron accumulations are typical hallmarks of brain ageing and several neurodegenerative disorders particularly PD. Numerous studies on post mortem tissue report on an increased amount of total iron in the substantia nigra in patients with PD also supported by large body of in vivo findings from Magnetic Resonance Imaging (MRI) studies. The importance and approaches for in vivo brain iron assessment using multiparametric MRI is increased over last years. Quantitative MRI may provide useful biomarkers for brain integrity assessment in iron-related neurodegeneration. Particularly, a prominent change in iron- sensitive T2* MRI contrast within the sub areas of the SN overlapping with nigrosome 1 were shown to be a hallmark of Parkinson's Disease with high diagnostic accuracy. Moreover, differential diagnosis between Parkinson's Disease (PD) and atypical parkinsonian syndromes (APS) remains challenging, mainly in the early phases of the disease. Advanced brain MR imaging enables to detect the pathological changes of nigral and extranigral structures at the onset of clinical manifestations and during the course of the disease. The Nigrosome-1 (N1) is a substructure of the healthy Substantia Nigra pars compacta enriched by dopaminergic neurons; their loss in Parkinson’s disease and atypical parkinsonian syndromes is related to the iron accumulation. N1 changes are supportive MR biomarkers for diagnosis of these neurodegenerative disorders, but its detection is hard with conventional sequences, also using high field (3T) scanner. Quantitative susceptibility mapping (QSM), an iron-sensitive technique, enables the direct detection of Neurodegeneration

Development and characterization of deep learning techniques for neuroimaging data

Deep learning methods are extremely promising machine learning tools to analyze neuroimaging data. However, their potential use in clinical settings is limited because of the existing challenges of applying these methods to neuroimaging data. In this study, first a data leakage type caused by slice-level data split that is introduced during training and validation of a 2D CNN is surveyed and a quantitative assessment of the model’s performance overestimation is presented. Second, an interpretable, leakage-fee deep learning software written in a python language with a wide range of options has been developed to conduct both classification and regression analysis. The software was applied to the study of mild cognitive impairment (MCI) in patients with small vessel disease (SVD) using multi-parametric MRI data where the cognitive performance of 58 patients measured by five neuropsychological tests is predicted using a multi-input CNN model taking brain image and demographic data. Each of the cognitive test scores was predicted using different MRI-derived features. As MCI due to SVD has been hypothesized to be the effect of white matter damage, DTI-derived features MD and FA produced the best prediction outcome of the TMT-A score which is consistent with the existing literature. In a second study, an interpretable deep learning system aimed at 1) classifying Alzheimer disease and healthy subjects 2) examining the neural correlates of the disease that causes a cognitive decline in AD patients using CNN visualization tools and 3) highlighting the potential of interpretability techniques to capture a biased deep learning model is developed. Structural magnetic resonance imaging (MRI) data of 200 subjects was used by the proposed CNN model which was trained using a transfer learning-based approach producing a balanced accuracy of 71.6%. Brain regions in the frontal and parietal lobe showing the cerebral cortex atrophy were highlighted by the visualization tools.

Induction of Hebbian associative plasticity through paired non-invasive brain stimulation of premotor-motor areas to elucidate the network's functional role

The ventral premotor cortex (PMv) is believed to play a pivotal role in a multitude of visuomotor behaviors, such as sensory-guided goal-directed visuomotor transformations, arbitrary visuomotor mapping, and hyper-learnt visuomotor associations underlying automatic imitative tendencies. All these functions are likely carried out through the copious projections connecting PMv to the primary motor cortex (M1). Yet, causal evidence investigating the functional relevance of the PMv-M1 network remains elusive and scarce. In the studies reported in this thesis we addressed this issue using a transcranial magnetic stimulation (TMS) protocol called cortico-cortical paired associative stimulation (ccPAS), which relies on multisite stimulation to induce Hebbian spike-timing dependent plasticity (STDP) by repeatedly stimulating the pathway connecting two target areas to manipulate their connectivity. Firstly, we show that ccPAS protocols informed by both short- and long-latency PMv-M1 interactions effectively modulate connectivity between the two nodes. Then, by pre-activating the network to apply ccPAS in a state-dependent manner, we were able to selectively target specific functional visuo-motor pathways, demonstrating the relevance of PMv-M1 connectivity to arbitrary visuomotor mapping. Subsequently, we addressed the PMv-to-M1 role in automatic imitation, and demonstrated that its connectivity manipulation has a corresponding impact on automatic imitative tendencies. Finally, by combining dual-coil TMS connectivity assessments and ccPAS in young and elderly individuals, we traced effective connectivity of premotor-motor networks and tested their plasticity and relevance to manual dexterity and force in healthy ageing. Our findings provide unprecedent causal evidence of the functional role of the PMv-to-M1 network in young and elderly individuals. The studies presented in this thesis suggest that ccPAS can effectively modulate the strength of connectivity between targeted areas, and coherently manipulate a networks’ behavioral output. Results open new research prospects into the causal role of cortico-cortical connectivity, and provide necessary information to the development of clinical interventions based on connectivity manipulation.

Pathogenetic mechanisms of gastrointestinal symptoms in a mouse model of Fabry disease: insights on the microbiota-gut-brain axis

Fabry disease (FD), X-linked metabolic disorder caused by a deficiency in α-galactosidase A activity, leads to the accumulation of glycosphingolipids, mainly Gb3 and lyso-Gb3, in several organs. Gastrointestinal (GI) symptoms are among the earliest and most common, strongly impacting patients’ quality of life. However, the origin of these symptoms and the exact mechanisms of pathogenesis are still poorly understood, thus the pressing need to improve their knowledge. Here we aimed to evaluate whether a FD murine model (α-galactosidase A Knock-Out) captures the functional GI issues experienced by patients. In particular, the potential mechanisms involved in the development and maintenance of GI symptoms were explored by looking at the microbiota-gut-brain axis involvement. Moreover, we sought to examine the effects of lyso-Gb3 on colonic contractility and the intestinal epithelium and the enteric nervous system, which together play important roles in regulating intestinal ion transport and fluid and electrolyte homeostasis. Fabry mice revealed visceral hypersensitivity and a diarrhea-like phenotype accompanied by anxious-like behavior and reduced locomotor activity. They reported also an imbalance of SCFAs and an early compositional and functional dysbiosis of the gut microbiota, which partly persisted with advancing age. Moreover, overexpression of TRPV1 was found in affected mice, and partial alteration of TRPV4 and TRPA1 as well, identifying them as possible therapeutic targets. The Ussing chamber results after treatment with lyso-Gb3 showed an increase in Isc (likely mediated by HCO3- ions movement) which affects neuron-mediated secretion, especially capsaicin- and partly veratridine-mediated. This first characterization of gut-brain axis dysfunction in FD mouse provides functional validation of the model, suggesting new targets and possible therapeutic approaches. Furthermore, lyso-Gb3 is confirmed to be not only a marker for the diagnosis and follow-up of FD but also a possible player in the alteration of the FD colonic ion transport process.

[the Use Of Fish On Buccal Smear To Investigate Mosaicism With A 45,x Cell Line: Study On Healthy Men And Patients With Disorders Of Sex Development].

To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.