Methylation profiling and validation of candidate tDMRs for identification of human blood, saliva, semen and vaginal fluid and its application in forensics.

Masters Degree. University of KwaZulu-Natal, Durban.Identification of body fluids and tissues is an essential step in forensic investigation because it can be used as strong evidence in identifying suspects and victims. Currently in forensic investigations, catalytic, enzymatic and immunological techniques are used to identify body fluids, however, are limited due to lack of sensitivity and specificity. Hence, researchers are always on the lookout for novel methods that can be used to identify and analyse body fluids. Recently, DNA methylation-based markers have proven to be more sensitive and specific than conventional methods for body fluid identification. Genome-wide methylation studies have demonstrated that tissue specific differentially methylated regions (tDMRs) vary in methylation profiles in various cell types and tissues. The differences in methylation profiles of tDMRs can be targeted to be used as biomarkers to differentiate between body fluids and tissues. To date, only a few DNA methylation-based markers have been reported to identify body fluids. To enhance the specificity and robustness of DNA methylation-based identification, novel markers are required. Additionally, methylation-based markers require further interrogation, to evaluate the stability of their methylation profiles under simulated forensics conditions such as UV light, temperature, rain and microbes, which could cause DNA degradation and affect DNA recovery as well as the methylation status of body fluids. In a previous study, based on differential gene expression in blood, saliva, semen and vaginal fluid, gene body CpG islands were selected, in genes Zinc finger protein 282 (ZNF282), Protein tyrosine phosphatase, receptor S (PTPRS) and Hippocalcin like 1 (HPCAL1), that have potential tDMRs to differentiate between, blood, saliva, semen and vaginal fluid. It was proposed that differential gene expression could be possibly due to differences in methylation patterns. The present study was undertaken to establish the methylation status of potential tDMRs in target body fluids by using methylation specific PCR (MSP) and bisulfite sequencing (BS). In both MSP and BS, the methylation status of 3 genes ZNF282, PTPRS and HPCAL1 were analysed in 10 samples of each body fluid. With MSP analysis the ZNF282 and PTPRS1 tDMR displayed semen-specific hypomethylation while HPCAL1 tDMR showed saliva-specific hypomethylation. The PTPRS 2 tDMR did not differentiate between any body fluids due to presence of methylation and unmethylation for all body fluids. With quantitative analysis by BS the ZNF282 tDMR showed statistically significant difference in overall methylation status between semen and all other body fluids as well as at individual CpG sites (p 0.05). The BS study showed that the tDMR for the HPCAL1 gene displayed non-specific amplification therefore was not further analysed. Furthermore, a sensitivity and forensic simulation study was conducted to determine the stability of methylation profiles. To determine the lowest DNA concentration that can be evaluated with MSP, a sensitivity study was conducted using five-fold serial dilution (25, 20, 15, 10, 5, 1 ng) of blood DNA samples. Each DNA dilution was subjected to bisulfite modification, followed by amplification with ZNF282, PTPRS 1, PTPRS 2, and HPCAL1 primers. The results showed that the detection limits were 10 ng for ZNF282 tDMR, 5 ng for PTPRS 1, 15 ng for PTPRS 2, and 5 ng for HPCAL1 tDMR. Thus, it was concluded that a DNA concentration greater than 10 ng would yield successful results with MSP analyses. To evaluate whether environmental conditions has an effect on the stability of methylation profiles of the ZNF282 tDMR, five samples of each body fluid were subjected to five different forensic simulated conditions (dry at room temperature, wet in an exsiccator, outside on the ground, sprayed with alcohol and sprayed with bleach) for 50 days. Following the 50 days, vaginal fluid showed highest DNA recovery under all conditions while semen had least DNA quantity. Under outside on the ground condition, all body fluids except semen showed decrease in methylation level, however, significant decrease in methylation level was observed for saliva. A statistical significant difference was observed for saliva and semen (p < 0.05) in the outside on the ground condition. No differences in methylation level were observed for the ZNF282 tDMR under all conditions for vaginal fluid samples. Thus, ZNF282 tDMR is stable under environmental insults and can be used as reliable semen-specific hypomethylated marker. The analysis of tDMRs represents a unique, efficient and reliable technique that can be used to differentiate between human body fluids. In the future, identification and validation of new tDMRs based markers as well as determining methylation differences in other forensically relevant body fluids will be beneficial for forensics applications.Supervisor on university system as Joshi, Meenu

Development of microfluidic applications to study the role of kainate receptors in synaptogenesis

Neurons have distinctive polar morphology with distinctive subcellular features comprising of cell soma, axons and dendrites. Primary objective of this study was to develop a novel microfluidic device for spatial isolation of axons from the somatodendritic compartment of cultured hippocampal neurons. A new method was developed for asymmetrical genetic manipulation improving specificity in studies of how individual proteins affect axonal morphology, presynaptic development and function. Subsequently, the microfluidic culture system was used to study the signaling events involved in synaptogenesis, focusing on the roles of kainate type of glutamate receptors (KARs). Functional studies have shown that KARs are present in axons and may regulate presynaptic function. However, the molecular composition and detailed subcellular localization of axonal KARs as well as their roles in presynaptic differentiation are largely unknown. The results show that axonal KARs promote early stages of synaptogenesis. Expression of low (GluK1-3) and high affinity (GluK4-5) KAR subunits promoted filopodiogenesis function at the isolated axons. In addition, axonal low affinity subunits enhanced clustering of synaptic vesicles and transmission efficacy at nascent glutamatergic synapses, an effect which was associated with widening of presynaptic active zone. High affinity KAR subunits had no effect on synaptic vesicle clustering, nor presynaptic transmission efficacy. However their heteromerization with low affinity subunits completely prevented the synapse promoting effects and instead lead to strong inhibition of presynaptic transmission efficacy. The presynaptic effects of GluK1-3 on synaptic vesicle clustering involved both PKA and PKC pathways. GluK1 expression was developmentally regulated in neonatal and juvenile hippocampus and heteromeric combination of GluK1c with high affinity subunits suppressed glutamatergic synaptic transmission. KARs are linked to various neurological and neuropsychiatric disorders. Our observations and previous findings strongly suggest that KARs are involved in morphological maturation of neurons and in refinement of neuronal circuitry in the brain. The present results provide novel insights into the involvement of different types of KAR subunits in synaptic development and morphological differentiation. Hence, they are potential therapeutic targets in various developmentally originating neurological disorders.Hermosolut ovat rakenteeltaan epäsymmetrisiä ja niillä on tyypillisesti yksi viejähaarake eli aksoni, jota pitkin hermosolu lähettää hermoimpulsseja. Hermoimpulssi välittyy kohdesoluun hermosolukontaktien eli synapsien kautta. Tämän työn tarkoituksena on ollut kehittää uusia menetelmiä aksonien ja aksoneissa sijaitsevien presynaptisten päätteiden erilaistumisen ja toiminnan tutkimiseksi. Työssä kehitettyjä mikrofluidisia soluviljelymenetelmiä on käytetty presynaptisten päätteiden kehitystä ohjaavien signaalimekanismien tutkimukseen. Erityisesti tutkimuksessa on pyritty selvittämään aksonaalisten kainaatti-tyypin glutamaattireseptorien (KAR) merkitystä synapsien kehityksessä. Tulokset osoittavat aksonaalisten kainaattireseptorien edistävän synapsien muodostumista hermosoluviljelmissä. Vaikutukset synapsien erilaistumiseen ja toimintaan olivat alayksikköriippuvaisia: kaikki KAR alayksiköt (GluK1-5) lisäsivät aksonaalisten filopodioiden muodostumista, mutta vain alayksiköt GluK1-GluK3 lisäsivät presynaptisten päätteiden muodostumista ja toimintaa. Nämä vaikutukset riippuivat proteiinikinaasien PKA ja PKC aktiivisuudesta. Lisäksi havaittiin KAR alayksikköjen GluK4 ja GluK5 estävän GluK1-3:n vaikutusta synapsien erilaistumiseen heteromeerisissa KAR komplekseissa. Työssä tutkittiin myös GluK1 alayksikön ilmenemistä ja toimintaa rotan hippokampuksessa varhaiskehityksen aikana. GluK1 ilmenemisen havaittiin olevan kehitysvaiheesta riippuvaa. Heteromeeristen GluK1 sisältävien KAR:n havaittiin estävän synaptista toimintaa hippokampuksen CA3 alueella. Kainaattireseptorit on liitetty erilaisiin neurologisiin ja neuropsykiatrisiin häiriöihin. Tämän työn tulokset vahvistavat käsitystä siitä, että kainattireseptoreilla on tärkeä merkitys hermoston kehityksen aikana ja tuovat kokonaan uutta tietoa eri KAR alayksiköiden vaikutuksista synapsien muodostumiseen. Tulokset edesauttavat uusien hoitomuotojen kehittämistä varhaiskehityksen aikana alkunsa saaville neurologisille sairauksille

Cerebrospinal fluid biomarkers in Alzheimer’s Disease: from bedside to bench and back

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that results in cognitive impairment and death. The pathological hallmarks are extracellular cortical amyloid plaques and intraneuronal tangles composed of hyperphosphorylated tau. Although environmental and genetic factors contribute to the development of AD, the sequence of pathophysiological events that lead to Alzheimer’s dementia is not yet completely clear. The clinical diagnosis of AD during life can be challenging and factors that explain clinical phenotypic heterogeneity and variability in rates of disease progression are not well understood. Biomarkers, objective measures of biological function, can be employed to support a clinical diagnosis of AD and may be abnormal before the onset of clinical symptoms. Imaging and cerebrospinal fluid biomarkers (CSF) are now incorporated into clinical and research diagnostic criteria. CSF, which is in direct contact with the brain, is a promising source of biomarkers and has the potential to differentiate AD from other neurodegenerative dementias, explain clinical heterogeneity within AD and elucidate the role of other pathobiological pathways. Ultimately CSF biomarkers might facilitate diagnosis of AD in its pre-clinical phase and allow for treatment responses to be measured. In this thesis CSF samples from clinical cohorts of individuals with AD, other neurodegenerative diseases and healthy controls are analysed using an extended panel of enzyme-linked immunosorbent assays (ELISA) and a novel mass spectrometry based assay. For the established CSF biomarkers, the practical issues related to collection, transportation and storage of CSF are investigated. Amyloid positron emission tomography (PET) imaging is investigated as a means of validating clinical cutpoints. An extended panel of established and emerging ELISAs is used to determine the diagnostic utility of biomarkers for differentiating AD from other neurodegenerative dementias and for explaining phenotypic heterogeneity within AD. The role of CSF biomarkers as predictors of disease progression is investigated employing robust measures of brain atrophy as surrogate measures of rates of neurodegeneration. Finally CSF samples are probed for new AD biomarkers using a novel mass spectrometry based assay. A number of practical conclusions are drawn from this work: aliquot storage volume is identified as an important confounder in measured CSF b-Amyloid concentration. CSF laboratory transportation methods are shown not to have a significant impact on measured biomarker concentration. Amyloid PET is a valuable means of validating clinical diagnostic cutpoints of core CSF biomarkers. Tau/Ab1-42 ratio, Ab40/42 ratio, P-tau and NFL emerge as having diagnostic utility for differentiating AD from other neurodegenerative diseases, and have high sensitivity and specificity for distinguishing AD from bvFTD, SD and healthy controls. Important differences in T-tau, P-tau and neurofilament light distinguish different AD atypical phenotypes and may help to elucidate underlying biological differences between these syndromes: individuals with the visual variant of AD (posterior cortical atrophy) have the lowest levels of CSF Tau and lowest rates of cognitive decline while the frontal executive cases have highest levels of NFL and highest rates of cognitive decline indicating more rapid neurodegeneration. Several novel biomarkers including trefoil factor 3 and several markers involved in vascular remodeling, amyloid processing and neuroinflammation are identified as predictors of increased atrophy rates in amyloid positive individuals suggesting possible independent mechanisms driving differing rates of neurodegeneration between individuals. Other novel AD biomarkers including malate dehydrogenase are identified as distinguishing AD from controls using a novel mass spectrometry based assay. Moreover, this assay demonstrates how mass spectrometry might be used for biomarker discovery and rapid development of a high throughput multiplexed clinical CSF assay. Taken together these results address some of the unanswered questions about how CSF should be collected, handled and stored to optimize analytical standardization, and how clinical results might be validated using amyloid PET. This work establishes the clinical utility of established biomarkers for differentiating AD from other neurodegenerative diseases and identifies established and novel biomarkers that might explain clinical heterogeneity and rates of progression between individuals. Finally a method for rapidly developing new biomarkers is tested and validated

Blood based biomarkers for the identification of Alzheimer’s disease using proteomics approaches

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease characterised by dysregulation of various cellular and molecular processes. Apart from environmental and lifestyle effects, genetic variations of the apolipoprotein E (APOE) gene plays a significant role in AD risk and progression, but these factors are poorly understood from a mechanistic perspective. In chapter 2, a meta-analysis of blood and CSF biomarkers of AD was performed, noting that the range of biomarkers studied has been restricted to a handful of classical proteins (Aβ and tau) and heavily focused on CSF. Still, more research is needed to establish robust blood tests to complement CSF or imaging tests for non-invasive testing options. Mass spectrometry significantly outperforms conventional antibody-based approaches such as ELISA and western blotting in specificity and quantification of low abundant proteins. Plasma proteomics has historically been limited by the lack of throughput and sensitivity, owing mainly to the complexity of the plasma samples. In chapter 3, I have developed a method for determining the fractionation strategy that provides in-depth plasma proteome coverage identifying 4,385 total proteins. This work demonstrates that simpler and faster approaches can provide substantial proteome coverage in conventional biochemistry laboratories. In chapter 4, I performed label-free proteomics analysis on plasma samples from clinical cohorts, using the newly developed fractionation method. Longitudinal and cross-sectional analyses of normal ageing and ageing with progression to MCI and AD were performed, based on plasma proteomic changes in the Sydney Memory and Ageing Study cohort. A replication cohort was used in chapter 5, the Australian Imaging, Biomarkers and Lifestyle study, which also included APOE e3 and e4 allele carriers. This additional information facilitated plasma proteome profiling to understand the impact of APOE e3 and APOE e4 carriers on AD dementia. Apart from comparing the effect of APOE genotypes on the AD proteome, I have confirmed a panel of reliable AD biomarkers that are consistently changing in both cohorts. In conclusion, I have successfully developed and applied MS-based fractionation methods for in-depth plasma proteome coverage of age, cognition and disease-related changes. Finally, a list of 44 plasma biomarkers consistently dysregulated in both AD cohorts presents a promising foundation for future clinical studies

Neuronal Network Oscillations in the Control of Human Movement

The overarching aim of this thesis was to use neuroimaging and neuromodulation techniques to further understand the relationship between cortical oscillatory activity and the control of human movement. Modulations in motor cortical beta and alpha activity have been consistently implicated in the preparation, execution, and termination of movement. Here, I describe the outcome of four studies designed to further elucidate these motor-related changes in oscillatory activity. In Chapter 3, I report the findings of a study that used an established behavioural paradigm to vary the degree of uncertainty during the preparation of movement. I demonstrate that preparatory alpha and beta desynchronisation reflect a process of disengagement from the existing network to enable the creation of functional assemblies required for movement. Importantly, I also demonstrate a novel neural signature of transient alpha synchrony, that occurs after preparatory desynchronisation, that underlies the recruitment of functional assemblies required for directional control. The study described in Chapter 4 was designed to further investigate the functional role of preparatory alpha and beta desynchronisation by entraining oscillatory activity in the primary motor cortex (M1) using frequency-specific transcranial alternating current stimulation. No significant effects of stimulation were found on participant response times. However, no clear conclusion could be drawn due to limitations of the stimulation parameters that were used. In Chapter 5, I explored the inverse relationship between M1 beta power and cortical excitability using single-pulse transcranial magnetic stimulation to elicit motor-evoked potentials (MEPs). The amplitude of MEPs collected during a period of beta desynchronisation was significantly greater than during a resting baseline. Conversely, the amplitude of MEPs collected during the post-movement beta rebound that follows the termination of a movement was significantly reduced compared to baseline. This finding confirms the inverse relationship between M1 beta power and cortical excitability. The study in Chapter 6 explored the effect of experimental context on M1 beta power. When the participant was cued to expect an upcoming motor task, resting beta power was significantly increased, then when the likelihood of an upcoming motor requirement decreased, there was a significant concurrent decrease in resting beta power. This reflects increased coherence and functional connectivity within M1 and other motor areas, to ‘recalibrate’ the motor system in preparation for a synchronous input signal to more readily recruit the required functional assembly

Amyloid beta: from pre-analytical factors to disease mechanisms

Background: Biomarkers are powerful tools for interrogating the basic science of disease processes, in the clinical detection of disease states, and as both targets and endpoints in therapeutic strategy. Amyloid beta (Aβ) is a core biomarker for Alzheimer’s disease (AD), but measurement variation between sites and experiments limits its potential. Furthermore, although the role of brain Aβ accumulation early in AD is extremely well attested, the biological mechanisms underlying this remain poorly understood. // Methods: To contribute to the development of treatments for AD patients and those at risk, this thesis set out to identify important pre-analytical confounding factors in Aβ measurement, strategies to mitigate them, and identify disease relevant patterns of Aβ peptide production in human CSF and an induced pluripotent stem cell-derived cortical neuron model of familial AD (fAD). // Results: A series of experiments demonstrated the importance of sample surface exposure to the measurement of Aβ peptides and tau. The volume at which samples are stored and iterative contact with fresh surfaces had profound effect on Aβ, but not tau, with greater surface exposure resulting in depletion of Aβ concentration. The mechanism was demonstrated to be protein surface adsorption. Importantly, the different Aβ peptides did not absorb to polypropylene to the same extent; Aβ42 concentration decreased proportionally more with surface exposure treatment than Aβ40 and Aβ38. It was observed that the addition of a non-ionic surfactant (Tween 20) to samples significantly mitigated the effect of surface exposure treatments on Aβ peptides and tau. However, the use of this additive did not meaningfully improve variability when sample storage conditions were standardised. Furthermore, variances in clinic to laboratory temperature and time interval did not significantly affect Aβ or tau concentration. Validation of an in vitro model of fAD was conducted. Experiment identified the use of Aβ ratios as a robust method for normalising data variability between and within cell lines over extended time periods. Furthermore, comparison of paired CSF, cell media, cell lysates, and post-mortem cortical tissue from the same individual demonstrated physiologically consistent patterns of Aβ ratios across sample types. Finally, comparison of multiple fAD mutation and control cell lines demonstrated quantitative and qualitative differences in secreted Aβ. APP V717I neurons increased secretion of Aβ42 and Aβ38 relative to Aβ43 and Aβ40. PSEN1 mutations increased secretion of longer Aβ peptides relative to shorter Aβ peptides, with mutation specific differences such as greatly increased Aβ43 in PSEN1 R278I.// Conclusions: This work demonstrated several novel considerations in the use of Aβ peptides as biomarkers for AD. Data principally highlight the importance of Aβ ratios to AD biomarker research, the necessity of controlling pre-analytical sample surface exposure intended for the measurement of ‘sticky’ protein biomarkers such as Aβ peptides, and the validity of iPSC-derived neuronal models for exploring the production of Aβ in AD and health

Determination of gene expression and proteome of brain tissue from schizophrenic patients

Orientador: Emmanuel Dias-NetoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A esquizofrenia é um distúrbio mental debilitante que afeta aproximadamente 1% da população mundial, caracterizado por sintomas produtivos como delírios e alucinações e sintomas negativos como apatia e decréscimo das emoções. Nesta tese, realizamos estudos do transcriptoma e do proteoma em tecido cerebral de pacientes com esquizofrenia, buscando identificar genes e proteínas envolvidas com esta doença. Em nossas análises transcricionais, utilizamos a técnica de Serial Analysis of Gene xpression (SAGE), uma abordagem ainda inédita em esquizofrenia. Os dados permitiram a análise de mais de 20 mil transcritos, e apontaram para o possível envolvimento de genes associados a processos como mielinização, função sináptica, metabolismo energético e homeostase de cálcio, incluindo genes anteriormente envolvidos com esquizofrenia, e também uma boa parcela de genes até então não associados com a doença. Uma pequena fração destes novos marcadores foi avaliada por Real-Time PCR permitindo a confirmação de alguns achados de SAGE. ossas análises de proteoma foram feitas com as técnicas de eletroforese de duas dimensões, seguida por espectrometria de massas e pela técnica de shotgun proteomics, também inédita em esquizofrenia. Estas análises foram realizadas com amostras de diferentes regiões cerebrais, incluindo córtex pré-frontal e lobo temporal anterior, e apontaram para alterações quantitativas em proteínas relacionadas com a homeostase de cálcio, citoesqueleto, metabolismo energético e de oligodendrócitos. De modo geral, observamos uma boa consistência entre os resultados obtidos quando estudamos diferentes classes de marcadores potenciais (genes e proteínas). Por muitas vezes os genes alterados não corresponderam a alterações quantitativas nas mesmas proteínas, no entanto, na maior parte dos casos, observamos alterações consistentes nas mesmas vias. Observamos a regulação diferencial do metabolismo de oligodendrócitos, energético, sináptico e revelamos a provável alteração da homeostase de cálcio em cérebros de pacientes com esquizofrenia, além de identificarmos genes e proteínas diferencialmente expressas nunca relacionadas à doença. Nossos dados reforçam achados prévios, apontam potenciais biomarcadores e podem fornecer novas pistas na compreensão da esquizofreniaAbstract: Schizophrenia is a mental debilitating disorder that affects 1% of the world population. It is characterized by positives symptoms such as delirium and hallucinations and negative symptoms such as apathy and emotion decrease. Here, we have studied the ranscriptome and proteome of brain samples of patients with schizophrenia, in an attempt to identify genes and proteins markers of the disease. Our transcriptome analyses of pre-frontal cortex were performed with Serial Analysis of Gene Expression (SAGE), here used for the first time in the study of schizophrenia. The data obtained allowed the analysis of approximately 20,000 transcripts, which suggested the importance of myelinization, synaptic function, energy metabolism and calcium homeostasis, in the genesis of schizophrenia. A series of genes previously implicated in the disease were identified, together with new potential markers which were revealed here for the first time. A small fraction of these was validated using realtime PCR, which confirmed some of the SAGE findings. Two-dimensional gel electrophoresis, mass spectrometry and shotgun proteomics were the approaches used here for large-scale protein analysis in schizophrenia. These approaches were used in brain samples derived from distinct areas such as pre-frontal cortex and anterior temporal lobe, and indicated quanitative alterations of proteins involved with calcium homeostasis, energy and oligodendrocyte metabolism and cytoskeleton. In general, a good correlation was observed when the different approaches (transcriptome and proteome) were used. In may cases, the alterations of some genes was not reflected by a correspondent alteration of the encoded protein. However, in most cases, reproducible alterations were found in the same pathways. Beside the identification of new schizophrenia-related genes and proteins, we also confirmed the the differential regulation of oligodendrocyte, synaptic and energetic metabolism, in this disease.Our data reinforce previous findings, and suggest new potential biomarkers that may contribute to the understanding of schizophreniaDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula