O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico. / Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.

<p><strong>SAMMANFATTNING</strong></p><p><strong>Syfte: </strong>Att beskriva förekomst av posttraumatisk stress och behov av känslomässigt stöd bland föräldrar till barn med hjärntumör vid två månader efter barnets diagnos (T2) respektive 12 månader efter avslutad cytostatika-/strålbehandling eller 18 månader efter operation av barn som inte fått cytostatika-/strålbehandling (T6). Syftet var också att jämföra behovet av känslomässigt stöd bland föräldrar med möjlig PTSD och hos de föräldrar som inte visar symtom på möjlig PTSD vid T2 respektive T6. <strong>Urval:</strong> 42 föräldrar till barn med hjärntumör deltog i studien: 20 mödrar och 22 fäder. <strong>Metod: </strong>Designen var deskriptiv longitudinell. Nivåer av PTSS och förekomst av möjlig PTSD mättes med PTSD Checklist Civilian, föräldrars behov av känslomässigt stöd mättes med ett studiespecifikt formulär. <strong>Resultat:</strong> Nivåer av PTSS hos föräldrar till barn med hjärntumör var lägre vid T6 än vid T2. Vid T2 hade 17 % möjlig PTSD jämfört med 5 % vid T6. Behovet av att samtala med psykolog var oförändrat lågt mellan T2 och T6, men behovet minskade över tid gällande att samtala med läkare, sjuksköterska, kurator, partner, vänner och övriga. <strong>Slutsats: </strong>Även om nivåer av PTSS och behovet av känslomässigt stöd på gruppnivå minskar över tid, bör vårdpersonal ändå uppmärksamma förälderns individuella behov av stöd. <strong></strong></p> / <p><strong>ABSTRACT</strong></p><p><strong>Aim:</strong> To describe occurrence of posttraumatic stress and the need of emotional support among parents of children with brain tumour two months after the child has been diagnosed (T2) respectively 12 months after completed chemo-/radiotherapy or 18 months after surgery of those children who did not receive chemo-/radiotherapy (T6). The aim was also to compare the need of emotional support between parents with occurrence of potential PTSD and parents who did not show any symptoms of potential PTSD at T2 and T6 respectively. <strong>Sample:</strong> 42 parents of children with brain tumour participated in the study: 20 mothers and 22 fathers. <strong>Method:</strong> The design was descriptive longitudinal. Levels of PTSS and occurrence of potential PTSD was measured by PTSD Checklist Civilian. Parents’ need of emotional support was measured with a study specific questionnaire. <strong>Results:</strong> Levels of PTSS was lower at T6 than T2. At T2 17 % had possible PTSD compared with 5 % at T6. The need to talk to a psychologist was unchanged low between T2 and T6, but the need declined over time concerning to talk with a doctor, nurse, welfare officer, partner, friends and others. <strong>Conclusion:</strong> Even if levels of PTSS and the need of emotional support in group level decline over time, nursing staff should still be ware of the parents’ individual need of support.</p>

SAMMANFATTNING Syfte: Att beskriva förekomst av posttraumatisk stress och behov av känslomässigt stöd bland föräldrar till barn med hjärntumör vid två månader efter barnets diagnos (T2) respektive 12 månader efter avslutad cytostatika-/strålbehandling eller 18 månader efter operation av barn som inte fått cytostatika-/strålbehandling (T6). Syftet var också att jämföra behovet av känslomässigt stöd bland föräldrar med möjlig PTSD och hos de föräldrar som inte visar symtom på möjlig PTSD vid T2 respektive T6. Urval: 42 föräldrar till barn med hjärntumör deltog i studien: 20 mödrar och 22 fäder. Metod: Designen var deskriptiv longitudinell. Nivåer av PTSS och förekomst av möjlig PTSD mättes med PTSD Checklist Civilian, föräldrars behov av känslomässigt stöd mättes med ett studiespecifikt formulär. Resultat: Nivåer av PTSS hos föräldrar till barn med hjärntumör var lägre vid T6 än vid T2. Vid T2 hade 17 % möjlig PTSD jämfört med 5 % vid T6. Behovet av att samtala med psykolog var oförändrat lågt mellan T2 och T6, men behovet minskade över tid gällande att samtala med läkare, sjuksköterska, kurator, partner, vänner och övriga. Slutsats: Även om nivåer av PTSS och behovet av känslomässigt stöd på gruppnivå minskar över tid, bör vårdpersonal ändå uppmärksamma förälderns individuella behov av stöd. / ABSTRACT Aim: To describe occurrence of posttraumatic stress and the need of emotional support among parents of children with brain tumour two months after the child has been diagnosed (T2) respectively 12 months after completed chemo-/radiotherapy or 18 months after surgery of those children who did not receive chemo-/radiotherapy (T6). The aim was also to compare the need of emotional support between parents with occurrence of potential PTSD and parents who did not show any symptoms of potential PTSD at T2 and T6 respectively. Sample: 42 parents of children with brain tumour participated in the study: 20 mothers and 22 fathers. Method: The design was descriptive longitudinal. Levels of PTSS and occurrence of potential PTSD was measured by PTSD Checklist Civilian. Parents’ need of emotional support was measured with a study specific questionnaire. Results: Levels of PTSS was lower at T6 than T2. At T2 17 % had possible PTSD compared with 5 % at T6. The need to talk to a psychologist was unchanged low between T2 and T6, but the need declined over time concerning to talk with a doctor, nurse, welfare officer, partner, friends and others. Conclusion: Even if levels of PTSS and the need of emotional support in group level decline over time, nursing staff should still be ware of the parents’ individual need of support.

The most common form of childhood cancer is Acute Lymphoblastic Leukemia (ALL). Patients treated for ALL may experience short- and long-term physiological and cognitive effects due to treatment. However, delayed emotional and behavioral effects in pediatric survivors, as well as risk-factors that may make them more susceptible to developing problems with psychological and behavioral functioning are less understood. Studies investigating pediatric survivors have demonstrated that negative emotional and behavioral late effects can and do occur (Hobbie et al., 2000; Buizer et al. 2006; Novakovic et al., 1996; Mulhern, Wasserman, Friedman, & Fairclough, 1989), and it has been purported that survivors experience higher rates of depression, anxiety and low self-esteem (Koocher, O’Malley, Gogan, & Foster, 1980; Kazak, 1994). Anxiety in particular, has been identified as one of the longest lasting psychological sequelae of cancer (Kazak, 1994). Still, the data on long-term psychological sequelae is mixed, with some studies suggesting healthy, long-term, psychological adjustment (Brown et al., 1992; Fritz, Williams & Amylon, 1988; Greenberg, Kazak, & Meadows, 1989). This pilot study attempted to investigate emotional and behavioral late effects of cancer as reported by survivors and their caregivers on the Behavior Assessment System for Children, Second Edition (BASC-2). This study also investigated potential risk factors that made it more likely to develop emotional and behavioral late-effects. This study hypothesized that females, those undergoing high intensity chemotherapy, and those starting chemotherapy at an earlier age, would report significantly more internalizing and externalizing problems. Analysis revealed significant differences in reporting of anxiety, depression, attention and hyperactivity symptoms combined based on the age when treatment started. No other significant findings were uncovered; however, in an effort to provide directions for future research, patterns in the data were examined by comparing overall means on BASC-2 subscales. For example, females reported more hyperactive symptoms than males. In general, individuals who started treatment at younger ages reported more difficulty with emotional and behavioral functioning. Additionally, males and females adaptive behavior fell within normal limits. Overall, no BASC-2 mean scores were in the at-risk or clinically significant range of impairment suggesting adequate emotional, behavioral and adaptive functioning overall. / text

O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico. / Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.

O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico. / Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.

Resistance to glucocorticoid (GC) therapy results in poor prognosis for acute lymphoblastic leukemia (ALL) patients. Utilizing a whole genome shRNA screen our lab identified several novel mechanisms of GC resistance. My thesis work established that an orphan nuclear receptor, the Estrogen Related Receptor Beta (ESRRB), is critical for induction of apoptotic genes following treatment with the GC dexamethasone. ESRRB has mostly been implicated in maintenance of pluripotency in mouse embryonic stem cells. We find that repression of ESRRB results in GC resistance in ALL and define ESRRB as a novel cooperating transcription factor in GC-induced gene expression. We also show that agonists to ESRRB synergize with dexamethasone and increase dexamethasone induced apoptosis in relapse ALL patient samples. Interestingly, our shRNA screen identified another factor important in stem cell maintenance: SOX2. While we originally hypothesized that ESRRB and SOX2 may cooperate in ALL, RNA-sequencing studies revealed that these factors mediate GC resistance by independent mechanisms. Our data define SOX2 as a repressor of key signaling pathways in ALL. Upon SOX2 knockdown, we observe activation of pro-survival gene expression including activation of the MAPK pathway, which has previously been implicated in GC resistance. MAPK activation may be explained by an increase in EGFR expression observed in Sox2 knockdown cells and GC resistant patients, suggesting EGFR inhibitors may re-sensitize patients to GCs. Overall my thesis work identifies mechanisms of GC resistance in ALL and utilizes these findings to define novel therapeutic strategies for GC resistant ALL patients.

This study aimed to establish a connection, if any, between perceptions of information disclosure about medical treatment and decisional conflict in bereaved parents of children with cancer. Decisional regret was an important theme in this exploration because decisional conflict strongly aligns with the propensity to mentally redo past events, thereby forming counterfactual alternatives to reality. People generate counterfactuals to hypothesize a more favorable outcome subsequent to a negative event or the death of a child as applicable to this study. A secondary objective was to investigate the potential influence of counterfactual processing and regret on the construct of self-acceptance: a phenomenon researchers have rarely studied in the population of interest. Study participants included parents who lost a child to cancer in the United States after participating in medical treatment prescribed by a licensed oncologist. Cluster and convenience sampling were employed to recruit 92 participants. Quantitative methods were used in obtaining data samples through validated instruments for each independent and dependent variable. The responses collected indicate that a perceived lack of information disclosure about treatment risks and efficacy, yield a positive influence on decisional conflict after the death of a child. Similarly, decisional conflict positively correlates with decisional regret, while the latter negatively correlates with self acceptance in the bereavement process. The research implications call for additional studies that further isolate factors that contribute to decisional conflict. This study advocates for decision making tools and collaborative processes that ensure parents are well informed and involved in making medical decisions from diagnosis through palliative care, if a cure is not possible.

A compound-heterozygous variant occurs when a child inherits a variant from each parent, with these variants occurring at a different position within the same gene and on opposite homologous chromosomes. These inherited variants may result in two nonfunctional versions of the same gene. Compound-heterozygous variants cannot be identified unless a patients' DNA sequence data is phased. Phasing is a computationally demanding process that requires the use of multiple software tools in order to determine which nucleotide was inherited from which parent. First, in Chapter 1, we review the literature to better understand what research has been conducted on the role of compound-heterozygous variants in pediatric cancers and what methods are being used to identify them. In Chapter 2, we develop a pipeline to make it easier for us and other researchers to phase and identify compound-heterozygous variants using VCF files from trios or individuals. We then use this pipeline in Chapter 3 to survey the prevalence of compound-heterozygous variants across 7 pediatric disease types. We show the importance of identifying compound heterozygous and what information would be missed if this variant type was not included in study design. In Chapter 4, we develop a software tool to phase trio data using a combination of Mendelian inheritance logic and an existing phasing software program. We show that our software tool increases the total number of variants that can be phased. Finally, in Chapter 5, we use phased data of three nuclear families, each family having one child with pediatric cancer, to evaluate the potential to use inherited genomic variants to inform diagnostic decisions. The work contained within this dissertation shows the importance of not overlooking compound-heterozygous variants when trying to identify potentially causal genes in pediatric disease. In addition, this work provides software tools that are openly available for other researchers to use; these tools make it easier to phase patient DNA sequence data and to identify compound-heterozygous variants.

La tumeur de Wilms (TW) est un cancer du rein retrouvé principalement chez les enfants âgés de 2 à 4 ans. Elle représente 90% des cancers pédiatriques du rein. Le taux de survie des TW est supérieur à 90%. Ce dernier est rendu possible grâce à une stratification des patients en fonction du risque de récidive. Les TW sont classées dans différents groupes de risque selon le stade, l’histologie, la taille de la tumeur et la perte d’hétérozygotie aux loci 1p et 16q. Deux régions sont importantes dans la génétique des TW, WT1 et WT2. WT1 est un gène qui code pour un facteur de transcription important dans différentes phases du développement rénal. Des anomalies de WT1 sont retrouvées dans certains syndromes humains tels que WAGR et Denys-Drash qui sont associés à l’émergence de TW. WT2 est un locus présent dans la région chromosomique 11p15 où une perte d'hétérozygotie conduit à une disomie uniparentale. Ceci entraîne un risque accru de TW dû à une surexpression de IGF2 qui est liée à la prolifération d’organes et de membres. Malgré ce portrait génétique, il n’existe pas de sous-groupes moléculaires qui permettent une classification des TW comme on observe chez d’autres cancers de l’enfant, notamment la leucémie. Nous proposons de déterminer une stratification de sous-groupes groupes moléculaire à l’aide d’une signature moléculaire basée sur des profils d’expression qui améliorait la classification des TW dans leurs groupes de risque adéquat. Pour vérifier cette hypothèse, nous avons analysé le transcriptome d’une cohorte de 130 patients atteint d’une TW. Grâce à un regroupement hiérarchique du profil d’expression des échantillons, nous avons identifié deux sous-groupes potentiels de TW. Un de ces sous-groupes est défini par une sous-expression de WT1 et une surexpression de gènes du début du développement musculaire chez les enfants et montrerait un risque plus fort de rechute. Ces résultats montrent que des outils de regroupement basés sur une signature moléculaire permettraient d’identifier des sous-groupes moléculaires chez les TW. / Wilms' tumor (TW) is a kidney cancer found mainly in children aged from 2 to 4 years old. It represents 90% of pediatric kidney cancers. The TW survival rate is over 90%. The latter is made possible by stratifying patients according to the risk of relapse. TW are classified into different risk groups according to stage, histology, tumor size and more recently the loss of heterozygosity at loci 1p and 16q. Two regions are important in the genetics of WT, WT1 and WT2. WT1 is a gene that encodes an important transcription factor in different phases of renal development. WT1 abnormalities are found in some human syndromes such as WAGR and Denys-Drash which are associated with the emergence of TW. WT2 is a locus present in the chromosomal region 11p15 where a loss of heterozygosity leads to a uniparental disomy. This leads to an increased risk of WT due to an overexpression of IGF2 which is linked to the proliferation of organs and members. Despite this genetic portrait, there are no molecular subgroups which allow classification of TW as observed in other childhood cancers, including leukemia. We propose to determine a stratification of WT using a molecular signature based on expression profiles in their proper risk group. To test this hypothesis, we analyzed the transcriptome of a cohort of 130 WT patients. The hierarchical clustering of the of the sample’s expressions profiles identified two potential WT subgroups. One of these subgroups can be described by a lower expression of WT1 and overexpression of genes for early muscle development in children and show a higher risk of relapse. These results show that clustering tools based on a molecular signature could allow treatment adjustment (i.e. precision medicine) and thus increase the survival rate.