The loss of expression of the fragile X mental retardation protein (FMRP) leads to fragile X syndrome. Fragile X syndrome is the most prevalent inheritable mental retardation. FMRP has two types of RNA binding domains, two K-homology domains and an arginine-glycine-glycine box domain, and is proposed to act as a translation regulator of specific mRNA. Despite extensive research, the mechanism by which FMRP loss leads to the fragile X syndrome remains unclear. Thus, there is high interest to produce sufficient quantities of pure recombinant FMRP for biochemical and biophysical studies of the protein function. However, the recombinant bacterial expression of FMRP has had limited success, and subsequent recombinant eukaryotic and in vitro systems may produce FMRP which is posttranslationally modified, as phosphorylation and arginine methylation have been shown to occur on FMRP. In this study, we have successfully isolated the conditions for recombinant expression, purification and dialysis of full-length FMRP using Escherichia coli, with a high yield. The expression of FMRP using E. coli renders the protein devoid of the posttranslational modifications of phosphorylation and arginine methylation, allowing for the further study of the direct effects of these modifications individually and simultaneously. Additionally, FMRP has been shown to undergo alternative splicing, with one of the splicing sites in close proximity to the FMRP domain shown to be involved in binding G quadruplex mRNA with high affinity and specificity. We have analyzed how naturally occurring truncations in the FMRP sequence affect its RNA binding affinity, by applying the expression, purification and dialysis process to the second and third longest FMRP isoforms, followed by subsequent analysis of the G quadruplex mRNA binding properties by fluorescence spectroscopy. Our results show that as FMRP gets truncated by alternative splicing, its mRNA binding affinity increases. To test a model we proposed for FMRP translation regulation activity, we developed a luciferase reporter gene construct that contains the G quadruplex structure in the mRNA 5���-untranslated region. Using luminescence spectroscopy to analyze luciferase translation, we showed that low levels of full-length FMRP reduces luciferase translation, and as the concentration of full-length FMRP increases the luciferase translation increases. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry; / PhD; / Dissertation;

The purpose of this study was to explore what contributes to resilience in females diagnosed with Fragile X Syndrome. Fragile X Syndrome can be defined as an inherited (genetic) condition that causes mental impairment, attention deficit and hyperactivity, anxiety and unstable mood, autistic behaviours, hyper-extensible joints, and seizures. I became aware of Fragile X Syndrome during my time as a live-in caretaker to an adolescent female who was diagnosed with Fragile X Syndrome. Because she coped with her disability so resiliently, I was encouraged to explore what contributes to resilience in females diagnosed with Fragile X Syndrome. I followed a qualitative approach, anchored in the interpretivist paradigm. This means that I tried to understand the resilience of females diagnosed with Fragile X Syndrome through the meanings that the participants in my study assigned to them. Furthermore, I worked from a transformative paradigm, which meant that I was interested in changing the traditionally negative ways in which females diagnosed with Fragile X Syndrome are seen. I followed a multiple case study approach, which included four case studies. I conveniently selected the first participant, but realised that convenience sampling was not very credible for a qualitative case study. An Advisory Panel was then used to purposefully recruit three more participants. In order to explore what contributed to their resilience, I made use of interviews, observations, and visual data collection. I also interviewed adults (e.g. parents, teachers and consulting psychologists) who were significantly involved in the lives of my participants. My findings suggest that resilience in females with Fragile X Syndrome is rooted in protective processes within the individual as well as within her family and environment. Because my findings do not point to one specific resource, my study underscores newer understandings of resilience as an Eco systemic transaction. Most of the resilience-promoting resources noted by the participants in my study as contributing to their resilience have been identified as resilience-promoting in previous studies. Although the themes that emerged in my study have been reported in resilience previously, I make a contribution to theory because I link traditional resilience-promoting resources to resilience in females diagnosed with Fragile X Syndrome. Peer support was previously reported as a resilience-promoting resource, but in my study I noticed that the main source of peer support came from peers who were also disabled. Furthermore, my study transforms how we see females diagnosed with Fragile X Syndrome. This transformation encourages communities and families to work together towards resilience in females diagnosed with Fragile X Syndrome. / Ph.D, North-West University, Vaal Triangle Campus, 2011

The Indonesian archipelago comprises more than 17,000 islands, inhabited by ~200 million people constituting more than 350 recognizable ethnic and tribal groups which can be classified into two broad ethno-linguistic groups [the Austronesian (AN) and non-Austronesian (NAN) speaking peoples] and 3 physical anthropology groups (Deutero Malay, Proto Malay and Papuan). The origins of these groups are of considerable anthropological interest. The anthropology of Indonesia is extremely complex and still controversial. The present populations of Indonesia show very great diversity. The data presented below result from an investigation of the Fragile X A syndrome and the size and distribution of alleles at fragile sites on the X chromosome among Javanese males with developmental disability (DD) and unselected males from 10 major Indonesian ethnic groups. The Fragile X syndrome is caused by expansion of a CGG trinucleotide repeat array in the 5' untranslated region of the FMR-1 gene at Xq27.3. Normal X chromosomes have between 6-54 CGG trinucleotide repeats, whereas premutation alleles have 55-230 and full mutation alleles more than 230 repeats. In a study of predominantly Caucasian males with intellectual disability, the prevalence of Fragile X syndrome is estimated to be approximately 1:4,000. FRAXE mental retardation syndrome is caused by an expansion of a GCC trinucleotide repeat in the 5'UTR of FMR2 gene located 600 kb telomeric to FMR1. The prevalence of FMR2 is 1-2 per 100,000 live births. FMR2 common alleles consist of 11-30 GGC repeats; intermediate alleles between 31-60 GCC repeats; premutation alleles with 61-200 repeats and full mutation alleles have over 200 repeats with attendant methylation of the repeat array The first Indonesian screening program aimed at determining the presence and prevalence of fragile XA syndrome among individuals with mild DD (IQ above 50) from special schools (N=205) and isolated areas (N=50) of Java was undertaken in 1994-1996 by cytogenetic and molecular studies. In this first study 4 fragile X positive children were found among 255 males with DD. The estimated prevalence of fragile-X in males with mild DD from special schools was 1.95% (5/205) and the overall prevalence was 1.57% (4/255). The number of trinucleotide repeats in the 5' untranslated regions of the FMR1 and FMR2 genes were determined by PCR in 254 Fragile XA-negative Javanese male children with DD. The distribution of FMR1 and FMR2 trinucleotide repeat alleles was found to be significantly different in the Indonesian population with DD compared to that in equivalent Caucasian populations. The trimodal distribution of Indonesian FMR1 alleles (29, 30 and 36 repeats) is largely in agreement with findings from other Asian populations). This provides supportive evidence that the origin of Indonesians could be the same as that of the Chinese and Japanese. Sequence analysis was performed on the trinucleotide repeat arrays of the 27 individuals' FMR1 alleles in the 'grey zone' (35-52 repeats). The identification of 16 unrelated individuals with a (CGG)36 allele that also contains a (CGG)6 segment [(CGG)9AGG(CGG)9AGG(CGG)6 AGG(CGG)9 or 9A9A6A9 pattern] is in agreement with earlier observations in the Japanese population. It is proposed that this FMR1 array pattern may be specific for Asian populations and that Javanese and Japanese populations may have arisen from a single progenitor population. The presence of pure 25, 33 and 34 CGGs in FMR1 alleles with 36, 44 and 45 repeats respectively, suggests that these may represent alleles at high risk for instability and may therefore be at early stages of expansion to a premutation. The lack of the characteristic (CGG)6 in all three alleles with ?? 25 pure CGG arrays suggests that the most common Asian 36 repeat allele is not predisposed to slippage expansion. Seven of the 8 alleles with 36 CGG repeats could be sequenced. Seven of 36 CGG repeats FMR1 alleles from the Hiri population has been sequenced and 4 alleles indicated 9A9A6A9 pattern, 1 sample with 10A25 pattern Two of the remaining alleles showed 12A6A6A9 structure, which consisted of a tandem duplication of the (CGG)6 segment. The presence of a tandem duplication of (CGG)6 segments has never been reported in any other population. The other major findings of this study are that FRAXE syndrome is a rare cause of developmental disability in this predominantly-Javanese population. The most common FMR2 (GCC)20 allele in this selected Asian population is significantly longer than that previously reported for Caucasian populations. There was a weak correlation between the overall length of the FMR1 and FMR2 repeat arrays within the normal range (Spearman's Rank Correlation = 0.130, p-value=0.042) in the Indonesian population, which have been no previous associations reported for alleles within the normal range. One approach to studying the origins of the human populations is to study the genetic structure of polymorphic alleles such as those at the FMR1 locus and its linked microsatellite markers DXS548 and FRAXAC1. Length polymorphisms of the FMR1 gene (CGG)n repeat array, DXS548 and FRAXAC1 were studied in a total of 1,008 unselected males from 10 different Indonesian ethnic groups. FMR1 alleles were identified ranging from 8 to 57 CGG repeats. The most common CGG repeat allele was 29 (45.6%) followed by 30 (27.4%) and 36 repeats (8.0%). One hundred and forty four grey zone (3-52 CGG) alleles were found in the study population. Four people of the same ethnic group from an isolated island in Eastern Indonesia (Hiri, Ternate), a representative of the NAN ethnolinguistic group, had CGG repeat lengths of 55-57. The prevalence of these alleles is estimated to be 3.3% (4/120) in the population of Hiri or 0.4% (4/1008) of whole Indonesian population. Thirteen different alleles were found at the DXS548 locus, of which allele numbers 7 [194 bp] (44.1%), 6.5 [195bp] (43.5%) and 6 [196bp] (7.5%) are the most common. Seven rare alleles, some of which have not been previously found in Asian peoples were also identified (190, 191,192, 193, 197,198, 199, 202, 204 and 206) and accounted for 3.9% of the total. The odd number alleles were dominantly found in this study whereas almost none found in Caucasian. The finding of many "odd numbered" alleles DXS548 has never been found in other Asian population and has only been documented extremely rarely in Caucasians and Africans. Five different alleles of FRAXAC1 identified with alleles D [106 bp] (62.2%) and C [108bp] (35.6%) accounting for 97.8% of FRAXAC1 alleles in the population. Three rare alleles (104, 110, 112 bp = 2.2%) were identified that have not been previously found in other Asian populations (1-3). There is a striking linkage disequilibrium of FMR1 alleles with FRAXAC1 (p=0.0001), 88% of 29 (CGG)n repeats alleles associated with FRAXAC1 allele D (106bp) versus only 17% with the 30 (CGG)n repeat alleles, which is in agreement with other studies. The value of D' was calculated to be 0.7. The longer alleles of both DXS548 and FRAXAC1 were found mostly in the NAN ethnolinguistic group. Moreover the Irian Jaya people also showed a higher percentage of people with 30 CGG repeats and the 108 bp FRAXAC1. The Eastern Indonesian NAN groups demonstrate a different genetic background probably due to the contribution of Melanesian peoples. The Analysis of Molecular Variance (AMOVA) identified that the vast majority of genetic diversity occurs within, rather than between, ethnic groups. These data are consistent with a model where there is sufficient migration (~20 per generation) between populations to minimise differentiation of population through genetic drift. The results obtained are consistent with three clusters of populations that share similar allele frequencies at the fragile X locus. The most clearly defined cluster is based in the east of Indonesia and includes the two Irian populations, Minahasans and Hiri. A surprising finding was that the Minahasan who are Deutero-Malay in origin and physical appearance are genetically closer to the Irianese. This may reflect the admixture of Melanesian alleles or other eastern Indonesian alleles as a result of their geographic location in that part of Indonesia. The second major cluster is largely based in the west of the country and is composed of the following Deutero-Malay populations; Javanese, Balinese, Acehnese but which also includes people from Ternate (not including those from Hiri). Using Delta Mu and Nei's genetic distance for FMR1 locus in this study the Javanese were shown to have the closest distance to Balinese which is consistent with anthropological data and with published data. The third group is a "western and central" group composed of Bimanese, Dayak and Sundanese who share some features of the western and eastern clusters but mostly resemble the western Indonesian populations. Bima is located in the lesser Sunda in between west Indonesia and east Indonesia. The Bimanese are of mixed Deutero & Proto Malay origin that is consistent with their geographic location. The Bataks are distinctive and sit somewhat apart in this scheme. In this study, Bataks were found not to resemble the other Proto-Malay group studied (the Dayak). The Dayaks were found to have fewer alleles than the Bataks at FRAXAC1 and DXS548. In all four methods of calculating genetic distance Bataks showed a large genetic distance to almost all other ethnic groups. There are differences in allele frequency between east and west Indonesia as well as other Asian nations, but the genetic similarities between these groups are also very impressive. The findings from this study are consistent with other genetic anthropological evidence that the people of Indonesia have the same origin as North-east Asian groups. This model is referred to as the "express train from Taiwan" in which the Austronesian speakers are proposed to have radiated from Taiwan bringing the Malayo-Polynesian language group to the Philippines, Borneo and Sulawesi around 5000-4500 B.P.E. However Richards et al.(1998) have used the diversity in the mtDNA D Loop to propose an alternative to the "express train" model. The "two train7quot; model proposes that the Austronesian languages originated within eastern Indonesia during the Pleistocene era and spread through Melanesia and into the remote Pacific within the past 6,000 years. Unfortunately the high migration rates between population groups that were demonstrated in this thesis and the known migration patterns of populations through Indonesia preclude determining whether the observed allelic heterogeneity is a function of the original population or due to the admixture of several gene pools in more recent times.

Contains copies of author's previously published articles. / Includes bibliographic references. / 1 v. various foliations : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1992

The Indonesian archipelago comprises more than 17,000 islands, inhabited by ~200 million people constituting more than 350 recognizable ethnic and tribal groups which can be classified into two broad ethno-linguistic groups [the Austronesian (AN) and non-Austronesian (NAN) speaking peoples] and 3 physical anthropology groups (Deutero Malay, Proto Malay and Papuan). The origins of these groups are of considerable anthropological interest. The anthropology of Indonesia is extremely complex and still controversial. The present populations of Indonesia show very great diversity. The data presented below result from an investigation of the Fragile X A syndrome and the size and distribution of alleles at fragile sites on the X chromosome among Javanese males with developmental disability (DD) and unselected males from 10 major Indonesian ethnic groups. The Fragile X syndrome is caused by expansion of a CGG trinucleotide repeat array in the 5' untranslated region of the FMR-1 gene at Xq27.3. Normal X chromosomes have between 6-54 CGG trinucleotide repeats, whereas premutation alleles have 55-230 and full mutation alleles more than 230 repeats. In a study of predominantly Caucasian males with intellectual disability, the prevalence of Fragile X syndrome is estimated to be approximately 1:4,000. FRAXE mental retardation syndrome is caused by an expansion of a GCC trinucleotide repeat in the 5'UTR of FMR2 gene located 600 kb telomeric to FMR1. The prevalence of FMR2 is 1-2 per 100,000 live births. FMR2 common alleles consist of 11-30 GGC repeats; intermediate alleles between 31-60 GCC repeats; premutation alleles with 61-200 repeats and full mutation alleles have over 200 repeats with attendant methylation of the repeat array The first Indonesian screening program aimed at determining the presence and prevalence of fragile XA syndrome among individuals with mild DD (IQ above 50) from special schools (N=205) and isolated areas (N=50) of Java was undertaken in 1994-1996 by cytogenetic and molecular studies. In this first study 4 fragile X positive children were found among 255 males with DD. The estimated prevalence of fragile-X in males with mild DD from special schools was 1.95% (5/205) and the overall prevalence was 1.57% (4/255). The number of trinucleotide repeats in the 5' untranslated regions of the FMR1 and FMR2 genes were determined by PCR in 254 Fragile XA-negative Javanese male children with DD. The distribution of FMR1 and FMR2 trinucleotide repeat alleles was found to be significantly different in the Indonesian population with DD compared to that in equivalent Caucasian populations. The trimodal distribution of Indonesian FMR1 alleles (29, 30 and 36 repeats) is largely in agreement with findings from other Asian populations). This provides supportive evidence that the origin of Indonesians could be the same as that of the Chinese and Japanese. Sequence analysis was performed on the trinucleotide repeat arrays of the 27 individuals' FMR1 alleles in the 'grey zone' (35-52 repeats). The identification of 16 unrelated individuals with a (CGG)36 allele that also contains a (CGG)6 segment [(CGG)9AGG(CGG)9AGG(CGG)6 AGG(CGG)9 or 9A9A6A9 pattern] is in agreement with earlier observations in the Japanese population. It is proposed that this FMR1 array pattern may be specific for Asian populations and that Javanese and Japanese populations may have arisen from a single progenitor population. The presence of pure 25, 33 and 34 CGGs in FMR1 alleles with 36, 44 and 45 repeats respectively, suggests that these may represent alleles at high risk for instability and may therefore be at early stages of expansion to a premutation. The lack of the characteristic (CGG)6 in all three alleles with ?? 25 pure CGG arrays suggests that the most common Asian 36 repeat allele is not predisposed to slippage expansion. Seven of the 8 alleles with 36 CGG repeats could be sequenced. Seven of 36 CGG repeats FMR1 alleles from the Hiri population has been sequenced and 4 alleles indicated 9A9A6A9 pattern, 1 sample with 10A25 pattern Two of the remaining alleles showed 12A6A6A9 structure, which consisted of a tandem duplication of the (CGG)6 segment. The presence of a tandem duplication of (CGG)6 segments has never been reported in any other population. The other major findings of this study are that FRAXE syndrome is a rare cause of developmental disability in this predominantly-Javanese population. The most common FMR2 (GCC)20 allele in this selected Asian population is significantly longer than that previously reported for Caucasian populations. There was a weak correlation between the overall length of the FMR1 and FMR2 repeat arrays within the normal range (Spearman's Rank Correlation = 0.130, p-value=0.042) in the Indonesian population, which have been no previous associations reported for alleles within the normal range. One approach to studying the origins of the human populations is to study the genetic structure of polymorphic alleles such as those at the FMR1 locus and its linked microsatellite markers DXS548 and FRAXAC1. Length polymorphisms of the FMR1 gene (CGG)n repeat array, DXS548 and FRAXAC1 were studied in a total of 1,008 unselected males from 10 different Indonesian ethnic groups. FMR1 alleles were identified ranging from 8 to 57 CGG repeats. The most common CGG repeat allele was 29 (45.6%) followed by 30 (27.4%) and 36 repeats (8.0%). One hundred and forty four grey zone (3-52 CGG) alleles were found in the study population. Four people of the same ethnic group from an isolated island in Eastern Indonesia (Hiri, Ternate), a representative of the NAN ethnolinguistic group, had CGG repeat lengths of 55-57. The prevalence of these alleles is estimated to be 3.3% (4/120) in the population of Hiri or 0.4% (4/1008) of whole Indonesian population. Thirteen different alleles were found at the DXS548 locus, of which allele numbers 7 [194 bp] (44.1%), 6.5 [195bp] (43.5%) and 6 [196bp] (7.5%) are the most common. Seven rare alleles, some of which have not been previously found in Asian peoples were also identified (190, 191,192, 193, 197,198, 199, 202, 204 and 206) and accounted for 3.9% of the total. The odd number alleles were dominantly found in this study whereas almost none found in Caucasian. The finding of many "odd numbered" alleles DXS548 has never been found in other Asian population and has only been documented extremely rarely in Caucasians and Africans. Five different alleles of FRAXAC1 identified with alleles D [106 bp] (62.2%) and C [108bp] (35.6%) accounting for 97.8% of FRAXAC1 alleles in the population. Three rare alleles (104, 110, 112 bp = 2.2%) were identified that have not been previously found in other Asian populations (1-3). There is a striking linkage disequilibrium of FMR1 alleles with FRAXAC1 (p=0.0001), 88% of 29 (CGG)n repeats alleles associated with FRAXAC1 allele D (106bp) versus only 17% with the 30 (CGG)n repeat alleles, which is in agreement with other studies. The value of D' was calculated to be 0.7. The longer alleles of both DXS548 and FRAXAC1 were found mostly in the NAN ethnolinguistic group. Moreover the Irian Jaya people also showed a higher percentage of people with 30 CGG repeats and the 108 bp FRAXAC1. The Eastern Indonesian NAN groups demonstrate a different genetic background probably due to the contribution of Melanesian peoples. The Analysis of Molecular Variance (AMOVA) identified that the vast majority of genetic diversity occurs within, rather than between, ethnic groups. These data are consistent with a model where there is sufficient migration (~20 per generation) between populations to minimise differentiation of population through genetic drift. The results obtained are consistent with three clusters of populations that share similar allele frequencies at the fragile X locus. The most clearly defined cluster is based in the east of Indonesia and includes the two Irian populations, Minahasans and Hiri. A surprising finding was that the Minahasan who are Deutero-Malay in origin and physical appearance are genetically closer to the Irianese. This may reflect the admixture of Melanesian alleles or other eastern Indonesian alleles as a result of their geographic location in that part of Indonesia. The second major cluster is largely based in the west of the country and is composed of the following Deutero-Malay populations; Javanese, Balinese, Acehnese but which also includes people from Ternate (not including those from Hiri). Using Delta Mu and Nei's genetic distance for FMR1 locus in this study the Javanese were shown to have the closest distance to Balinese which is consistent with anthropological data and with published data. The third group is a "western and central" group composed of Bimanese, Dayak and Sundanese who share some features of the western and eastern clusters but mostly resemble the western Indonesian populations. Bima is located in the lesser Sunda in between west Indonesia and east Indonesia. The Bimanese are of mixed Deutero & Proto Malay origin that is consistent with their geographic location. The Bataks are distinctive and sit somewhat apart in this scheme. In this study, Bataks were found not to resemble the other Proto-Malay group studied (the Dayak). The Dayaks were found to have fewer alleles than the Bataks at FRAXAC1 and DXS548. In all four methods of calculating genetic distance Bataks showed a large genetic distance to almost all other ethnic groups. There are differences in allele frequency between east and west Indonesia as well as other Asian nations, but the genetic similarities between these groups are also very impressive. The findings from this study are consistent with other genetic anthropological evidence that the people of Indonesia have the same origin as North-east Asian groups. This model is referred to as the "express train from Taiwan" in which the Austronesian speakers are proposed to have radiated from Taiwan bringing the Malayo-Polynesian language group to the Philippines, Borneo and Sulawesi around 5000-4500 B.P.E. However Richards et al.(1998) have used the diversity in the mtDNA D Loop to propose an alternative to the "express train" model. The "two train7quot; model proposes that the Austronesian languages originated within eastern Indonesia during the Pleistocene era and spread through Melanesia and into the remote Pacific within the past 6,000 years. Unfortunately the high migration rates between population groups that were demonstrated in this thesis and the known migration patterns of populations through Indonesia preclude determining whether the observed allelic heterogeneity is a function of the original population or due to the admixture of several gene pools in more recent times.

Thesis (Ph. D.)--Dept. of Paediatrics, Faculty of Medicine, University of Adelaide, 1991. / Typescript (Photocopy). Includes published papers co-authored by the author at the end of volume 2. Includes bibliographical references (leaves 195-237 of vol. 1).

Thesis (Ph. D.)--University of New South Wales, 1998. / Also available online.

Fragile X Syndrome (FXS) is a disorder caused by a congenital mutation of the FMR1 gene on the X chromosome. FXS is associated with moderate to severe intellectual disability and is one known cause of autism spectrum disorders. There are no approved medications to treat FXS symptoms. In 2013, Seaside Therapeutics completed two Phase 3 studies of an investigational medication, STX209, for treatment of social withdrawal in FXS. Efficacy results for these studies were not positive. Clinical trials of psychoactive drugs often fail to show a statistical difference from placebo controls and a robust response to placebo is often cited as a reason for the failure. Retrospective studies of baseline variables in clinical trials have identified characteristics that were associated with an increased likelihood of responding to placebo. Such information is valuable for the design of future clinical trials and no such studies have been conducted in FXS. This study was a post-hoc analysis of data from Seaside Therapeutics' Phase 3 clinical trials in FXS. Baseline variables for subjects receiving placebo were pooled for analysis. To determine if a subject responded to placebo, the parent-rated ABC-SA and the ABC-IR were used. Clinician-rated assessments, including the CGI-S and CGI-I, were examined as well. Two-sample t-testing, one-way ANOVA testing, and correlation coefficients were calculated to compare the responses of subjects with different baseline characteristics. General linear regression modeling was used to determine if there were multiple baseline variables that could predict placebo response. Logistic regression modeling was used to determine if the baseline variables could predict whether a subject had a higher chance of being a treatment responder. A total of 287 subjects were randomized and completed the Phase 3 studies. Analyses for this study were conducted in a subgroup containing 106 subjects who received placebo. 76% improved during the study on the ABC-SA, indicating that there was a strong placebo effect on the study. None of the dichotomous baseline variables were associated with statistically significant differences in ABC-SA, ABC-IR, CGI-S, or CGI-I scores. Placebo-treated subjects in the 209FX302 study who were taking antipsychotics improved less on the CGI-S than those not on those medications. A similar pattern was observed on the ABC-IR and ABC-SA. Other categorical baseline variables were tested and there was no difference in the mean changes. The CGI-S score at baseline appeared to predict a statistically significant difference in the ABC-IR as more severe subjects were more likely to show a larger change in the ABC-IR. Similar, although not statistically significant results were seen with ABC-SA, CGI-I, and CGI-S changes, in that more severe subjects had greater responses to placebo. ABC-IR score changes were correlated independently with each of the ABC-C subscales but also with parental distress, CGI-S, and VAS-Anxiety. Only one variable, the ABC-IR at baseline, was significantly correlated with the ABC-SA score change, the rest of the variables were not significant. A multiple linear regression model predicting placebo response for the ABC-SA included only the baseline ABC-SA score. When the studies were modeled separately, the 209FX302 model contained additional variables including gender, antipsychotic use, and ABC-stereotypy scores. For the ABC-IR change model, the highest correlation coefficient was found in the 209FX301 study with ABC-IR, gender, Vineland-communication, maternal FMR1 status, and ABC-SL included in the model. 70% of the placebo treated subjects improved on the ABC-SA by at least 25%. Placebo responders were less frequently observed in clinician-rated assessments such as the CGI-I and CGI-S. In logistic regression models, for the ABC-IR response, a higher score on the hyperactivity subscale of the ABC-C was predictive of a lower placebo response. The CGI-S model was statistically significant and included the subject's age, race and ABC-IS score. The ABC-SA response could be modeled only in the 209FX302 study with gender and ADHD medication use remaining in the model. Also in the 209FX302 study, subjects were far less likely to be a responder on the ABC-IR or a total responder, if they were taking antipsychotic medications. Results of this study indicate that the ABC-SA is not recommended in future trials in the FXS patient population. Future trials should also allow ADHD and antipsychotic medication use as they were associated with a lower placebo response in some analyses. In addition, due to their inclusion in regression models, future studies should consider baseline variables such as parental stress and Vineland scores, and when designing study eligibility criteria or stratification variables.

Resumo: A síndrome do cromossomo X frágil (SXF) é a forma mais comum de deficiência mental herdada. A doença ocorre pela expansão das repetições de trinucleotídeos na região 5' não traduzida do gene FMR1 no cromossomo X. Dependendo do número de repetições CGG originam-se 4 tipos de alelos: normal (NL), pré-mutado (PM), gray zone (GZ) e mutação completa (FM). A instabilidade e expansão das repetições, aliado à metilação do DNA, causam a diminuição ou ausência na produção da proteína FMRP, a qual é essencial para a função cerebral. O diagnóstico da SXF tem sido realizado principalmente por análise molecular Southern blot. Porém, este método é trabalhoso, demorado e de custo elevado. Recentemente foi desenvolvido um novo método molecular para triagem da SXF por PCR, que segundo os autores, é rápido, de baixo custo, e eficiente na detecção das repetições CGG em homens e mulheres. No entanto, notou-se a ausência de informações importantes para reprodução do método. Os objetivos deste estudo foram: (i) padronizar a técnica de PCR proposta por Tassone et al., (2008), adaptando-a, devido a carência de informações metodológicas; (ii) comprovar a exatidão (acurácia), sensibilidade e especificidade do método, comparando-a ao Southern blot; (iii) avaliar a aplicação da técnica utilizando DNA extraído de diferentes materiais biológicos/métodos de extração; (iv) estimar o custo e o tempo de execução do método no mercado nacional. Os materiais biológicos utilizados foram: sangue coletado por sistema à vácuo e células da mucosa oral, que foram extraídos por solventes orgânicos e sangue coletado em cartões FTA, purificado pelo kit Whatman. Obtevese sucesso na reprodução do método da PCR em 75 indivíduos utilizando a enzima Expand Long Template PCR System (Roche Diagnostics). A exatidão (acurácia), sensibilidade e especificidade foram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. The disease occurs by the expansion of triplet nucleotide repeats in the 5' untranslated of the FMR1 gene on chromosome X. Depending on the number of CGG repeats four types of alleles originate from it: normal (NL), pre-mutated (PM), gray zone (GZ) and full mutation (FM). The instability and expansion of these repetitions, together with the methylation of DNA, cause a decrease or absence in the production of the protein FMRP, which is essential for the brain function. The diagnosis of FXS has been done mainly by molecular analysis Southern blot. However, this method is laborious, time consuming and expensive. Recently we have developed a new molecular method for FXS screening by PCR, which according to the authors, is rapid, inexpensive, and efficient in the detection of CGG repeats in male and female. However, we noted the absence of important information for breeding method. The objectives of this study were: (i) to standardize the PCR technique proposed by Tassone et al. (2008), adapting it, due to the lack of methodological information, (ii) verify the accuracy, sensitivity and specificity of the method, comparing it to the Southern blot, and (iii) to evaluate the technique using DNA extracted from different biological materials / extraction methods, and (iv) estimate the cost and time of the method execution in the domestic market. The biological materials used were: blood collected by vacuum system and oral mucosal cells, which were extracted by organic solvents and blood collected on FTA cards, purified by Whatman kit. Success was achieved in the reproduction of the PCR method in 75 individuals using the enzyme Expand Long Template PCR System (Roche Diagnostics). The accuracy, sensitivity and specificity were 100% when analyzing the total sample, indicating that the technique can detect the presence... (Complete abstract click electronic access below) / Orientador: Regina Maria Barretto Cicarelli / Coorientador: Raquel Mantuaneli Scarel Caminaga / Banca: Robson Francisco Carvalho / Banca: Débora Aparecida Rodrigueiro / Mestre

The genetic causes of intellectual disability (ID) and autism spectrum disorder (ASD) are frequently associated with mutations in genes that encode synaptic proteins. A recent screen of ID patients has revealed that approximately 4% of individuals carry spontaneous autosomal-dominant de novo mutations in the SYNGAP1 gene. This gene encodes the synaptic GTPase activating protein (SYNGAP) a known regulator of Ras signalling. Investigations into the pathological consequences of Syngap1 haploinsufficiency (Syngap+/−) in mice have reported abnormalities in behaviour, synaptic plasticity and dendritic spine development. These are analogous to findings from the mouse model of fragile X syndrome (FXS; Fmr1-/y), the most common inherited form of ID. One of the prominent phenotypes reported in the mouse model of FXS is that a form of hippocampal long-term depression (LTD) mediated by the activation of Group 1 (Gp1) metabotropic glutamate (mGlu) receptors is enhanced and independent of new protein synthesis (Huber et al. 2002; Nosyreva et al. 2006). The cause of these synaptic plasticity deficits together with other cognitive abnormalities observed in FXS are thought to arise, in part, from excessive protein synthesis, the consequence of altered mGlu5 receptor signalling via the Ras-ERK1/2 signalling pathway. Enhanced protein synthesis rates in Fmr1-/y mice can be corrected by either inhibiting mGlu5 receptors or reducing Ras and subsequent ERK1/2 activity (Osterweil et al. 2013). In this thesis mGluR-dependent LTD was examined at Schaffer collateral/commissural inputs to CA1 pyramidal neurones in hippocampal slices obtained from Fmr1-/y, Syngap+/− and Fmr1-/ySyngap+/− double mutant mice. Extracellular field recordings reveal that acute application of the Gp1 mGluR agonist dihydroxyphenylglycine (DHPG) induces a form of mGluR-dependent LTD that is enhanced and independent of new protein synthesis in CA1 of Fmr1-/y mice. In Syngap+/− mice, the magnitude of mGluR-dependent LTD is also significantly increased relative to WT littermates and insensitive to protein synthesis inhibitors. Furthermore, in the Fmr1-/ySyngap+/− double mutant, Syngap haploinsufficiency occludes the increase in mGluR-dependent LTD caused by the loss of FMRP. In addition, metabolic labelling studies reveal basal protein synthesis rates to be modestly enhanced in the hippocampus of Fmr1-/y mice compared to WT mice. Importantly this phenotype translates to the rat model of FXS. In Syngap+/- hippocampal slices, basal protein synthesis rates are also significantly elevated compared to WT counterparts. Interestingly, elevated basal protein synthesis rates in Syngap+/- mice could be corrected in the hippocampus by similarly pharmacological strategies employed in Fmr1-/y mice. The comparable neuropathophysiology we observe between Syngap+/− and Fmr1-/y mice suggests that SYNGAP and fragile X mental retardation protein (FMRP) may converge on similar biochemical pathways raising the intriguing possibility that therapeutic strategies used in the treatment of FXS may also be of benefit in treating individuals with ID caused by mutations in SYNGAP1.