Dissertations / Theses on the topic 'Molecular probes Diagnostic use'

Pancreatic ductal adenocarcinoma (PDAC) has the poorest five-year survival rate of any cancer. Currently, there are no effective diagnostics or chemotherapeutics. Surgical resection is the only curative therapy. However, most patients experience recurrence due largely to challenges in assessing tumor margin status in the operating room. Molecular probes that selectively highlight pancreatic cancer tissue, having the potential to improve PDAC margin assessment intraoperatively, are urgently needed. In this work, a series of red and near-infrared fluorescent probes is reported. Two were found to distribute to normal pancreas following systemic administration. One selectively accumulates in genetically modified mouse models of PDAC, providing cancer-specific fluorescence. In contrast to the small molecule probes reported previously, it possesses inherent affinity for PDAC cells and tissue, and thus does not require conjugation to targeting agents. Moreover, the probe exhibits intracellular accumulation and enables visualization of four levels of structure including the whole organ, tissue, individual cells and subcellular organelles. It can thus promote new strategies for precision image-guided surgery, pancreatic cancer detection, the monitoring of therapeutic outcomes and basic research.

The purpose of this research was to study the hybridization of molecular beacons under different conditions and designs. Data collected suggest that the inconsistency found in the emission intensity of several of these probes may be caused by 3 important factors: length of the probe, nucleotide sequence and, the formation of an alternative complex structure such as a dimer. Of all three factors, dimer formation is the most troublesome, since it reduces the emission of the reporter molecules. A new probe design was used to reduce dimer formation. The emission signal of the improved probe was several folds stronger than those probes with the early design. In this research, dimer formation is detected, furthermore a new probe with a different design was tested. If dimer formation can be reduced molecular beacons can be integrated into more complex hybridization systems providing an important tool in research and diagnosis of genetic disorders.

The increasing incidence of disseminated (invasive) candidiasis is probably attributable to iatrogenic factors and to improved pre and postmortem evaluation. Premortem diagnosis of such infections have seldom been made early enough for successful treatment. In order to increase the likelihood of successful antifungal chemotherapy, rapid diagnosis of such infections is vital. However, present diagnostic procedures for invasive candidiasis are insensitive and often do not reliably differentiate superficial from invasive infections. This study was undertaken to produce DNA probes and to optimize conditions for rapid and efficient detection of Candida DNA. Seven random Candida albicans DNA fragments (2-7 kbp) were cloned into plasmid pACYC 184. These recombinant plasmids were labeled with either ³²p or biotin and used as probes. Two of the four recombinant plasmids tested were genus specific. The other two were slightly cross reactive with other yeasts (Saccharomyces cerevisiae and Hansenula anomala). Probes labeled with ³²p were twice as sensitive as the biotin probes. One ³²p labelled recombinant (#66) detected 7 Pg of target DNA , which corresponds to approximately 2 X 10⁵ C.albicans cells. With refined simple DNA extraction procedures for C.albicans (in serum), these recombinant probes could possibly be suitable for clinical application.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

This joint PhD program is part of a collaboration between Hong Kong Baptist University (Dr. Gary K-L Wong) and Laboratoire d'Ingénierie Moléculaire Appliquée à l'Analyse (LIMAA - Dr. Loïc Charbonnière) funded by the Alsace region to synthesize new nanoprobes for sensing, imaging, and inhibiting cancer diseases. The first work was to synthesize new hybrid ultrabright nanoparticles. They have been obtained from a La0.9Tb0.1F3 core and coated by different ligands. Thanks to a mechanism of antenna effect, the brightness of the nanoparticles has been significantly improved. The second work was to synthesize a new ligand to photosensitize water-soluble La0.90Eu0.1F3 nanoparticles in order to improve the emission of europium. A second ligand and new heterometallic nanoparticles have been synthesized with the aim to promote the energy transfer from Tb(III) ions on the surface of the NPs to Eu(III) ions in the core of the nanoparticles and to get a very long excited-state lifetime and an exceptional quantum yield in aqueous solution. The last work was to functionalize water-soluble graphitic-carbon nitride (g-C3N4) nanoparticles by porphyrins. The porphyrins have been synthesized to generate singlet oxygen (1O2), to host a Ga3+ ion inside their cavity and with two different linkers to be coupled to nanoparticles. This system aims to be a pH sensor, and a PDT and PET theranostic agent.

As new therapeutic targets and drugs are discovered for B-cell lymphoma and other cancers, companion diagnostics are also needed to determine target engagement, therapeutic efficacy, and patient segmentation for clinical trials. We first employed synthetic chemistry to build a platform for modifying small molecule drugs into imaging probes, using the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor AZD2281 (Olaparib) as a model for technology development. Our results showed that small-molecule companion imaging drugs can be used for fluorescence imaging in cells, as well as for pharmacokinetic studies and positron emission tomography (PET) imaging in vivo, without significantly perturbing their target binding properties or cellular uptake. To apply this approach to B-cell lymphoma drugs currently in clinical trials, we modified an irreversible inhibitor of Bruton's Tyrosine Kinase (BTK), PCI-32765 (Ibrutinib), with the fluorophore Bodipy FL (BFL), and used it for imaging in cells and in a mouse window-chamber xenograft model. The excellent co-localization of our probe (Ibrutinib-BFL) with BTK demonstrated its utility for studying additional BTK inhibitors and as a companion imaging probe. In parallel, we hypothesized that central nervous system (CNS) lymphoma diagnosis from paucicellular cerebrospinal fluid (CSF) samples could be improved with molecular profiling of putative lymphoma cells trapped in a customized microfluidic chip. Following fabrication and characterization of a polydimethylsiloxane (PDMS) diagnostic device containing an array of affinity-free single-cell capture sites, we were able to efficiently recover >90% of lymphocytes, perform immunostaining on chip, and apply an image-processing algorithm to group cells based on their molecular marker expression, such as kappa/lambda light chain restriction. Additionally, in combination with Ibrutinib-BFL or other imaging drugs, we demonstrated the potential for on-chip drug imaging for use in conjunction with drug development. Finally, we applied bioorthogonal conjugation chemistries on cellulose paper for potential applications in lowering the cost of drug screening. We anticipate that these approaches will enable direct, molecular information for personalized treatment decisions in B-cell lymphomas, as well as provide a roadmap for the development of companion diagnostic probes and devices for additional indications.

Increased blood plasma concentrations of the aminothiol homocysteine (Hcy) are associated with a variety of disease states including those which cause impaired renal function, many forms of cardiovascular disease, and neurodegenerative diseases such as Alzheimer's. Therefore, Hcy has the potential to be a significant diagnostic biomarker. Routine monitoring of Hcy plasma concentration is encumbered by the time and resources required to quantify Hcy using currently accepted instrumental analysis methods. As part of the continuing effort to develop a quick, reliable, inexpensive, and user-friendly test to quantify Hcy at the point of care, we have designed a series of novel colorimetric and fluorescent chemical probes based on bridged viologen structures. The absorbance at 540 nm for the para-bridged bis-nitrile viologen probe (pCN) was found to be proportional to the concentration of Hcy analyte, with LOD = 2.17 μM and LOQ = 6.10 μM where unhealthy Hcy plasma concentrations are > 15 μM. The mechanism of reactivity between pCN and Hcy encompasses a dynamic set of reactions which involve pimerization of radical probe species and thioether adduct formation of pCN with Hcy. Preliminary results with fluorometric analogs of the bridged viologen probes are also presented.

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006.
Dr. X. Hu, Committee Member ; Dr. Al Merrill, Committee Member ; Dr. Niren Murthy, Committee Member ; Dr. Gang Bao, Committee Chair ; Dr. Nicholas Hud, Committee Member. Includes bibliographical references.

Thesis (M.S.)--Biomedical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Bao, Gang; Committee Member: Merrill, Alfred; Committee Member: Santangelo, Philip. Part of the SMARTech Electronic Thesis and Dissertation Collection.

The most abundant biological thiols, homocysteine (Hcy), cysteine (Cys) and glutathione (GSH) have been the subject of intense research due to their association with a wide range of diseases. They play a key role in maintaining the redox status of biological systems. Selective detection methods for these thiols are challenging due to their similar structures and properties. Current commercially available detection methods use separations, fragile and expensive enzymatic or immunogenic materials and complex instrumentation. This has led to a global effort towards developing simple and inexpensive optical probes and indicators selective for specific biological thiols. Highly selective chemical probes and simple methods for detection and potential quantification of Hcy and GSH in their natural biological media have been developed. These indicators and methods are relatively simple and inexpensive for potential application at point of care. The selective detection of Hcy using novel asymmetric viologen chemical probes at room temperature is described as well as the use of commercially available materials under photochemical conditions. These probes respond linearly proportional to increasing Hcy concentrations, potentially enabling the monitoring of Hcy levels in human plasma. Additionally, new methods for the selective determination of GSH in human plasma, as well as its quantification in whole blood deposited on filter paper (dried blood spots), is also presented herein.

Giardia is the most common human parasite infection in the United States causing a lengthy diarrhea. Transmission of Giardia is by the fecal-oral route and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia in drinking water through the "Surface Water Treatment Rule." Current methods for detection of Giardia in water rely primarily on microscopic observation of water concentrates by immunofluorescent techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 base pairs long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of M13 vector with insert was isolated from lysed host E. coli XL1- Blue and used for production of the cDNA probe by nick translation with ³²P-labeled nucleotides. Seven different protocols were tested for extracting nucleic acids from the cysts. Using the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates by dot-blot hybridization assays. Environmental concentrates from secondary and tertiary treated sewage or surface waters were screened for Giardia cysts by immunofluorescent and the genespecific probe. Positive signals were observed in sewage and surface water samples without floatation at ten fold greater dilutions than after floatation. It appeared that gene probe detection was slightly more sensitive than microscopic detection of Giardia cysts for wastewater samples. In six surface water samples and two sewage sample no positive results were found either by the cDNA probe or immunofluorescent. Usually, DNA probes are radiolabeled and the most commonly used is ³²P. ³²P is expensive, hazardous and has an extremely short half-life of 14.3 days, necessitating frequent preparation of the nucleic acid probes. Three non-radioactive labeling methods, chemiluminescence, enzyme-linked immunoassay and enhanced chemiluminescence were evaluated. The cDNA probe was labeled by nick translation for chemiluminescence method. Biotinylated deoxyuridine triphosphate was used in place of deoxythymidine triphosphate to produce biotinylated DNA strands. The result of hybridization was visualized by chemiluminescenct detection of DNA. The sensitivity of the chemiluminescent method and the 32P labeled probe was 0.1 pg of DNA in a slot-blot hybridization assay.

Abstract Xenon atoms and sulfur hexafluoride (SF6) molecules can be dissolved in liquids and liquid crystals or adsorbed in porous materials. Nuclear magnetic resonance (NMR) spectra of 129Xe or 19F nuclei reveal information about their surroundings. This means that xenon atoms and SF6 molecules can be used as probes to indirectly study materials by NMR spectroscopy. The change in the spectra arises from a NMR interaction called shielding. Especially in the case of xenon, shielding reveals even the slightest changes, for example, in the density of a liquid it is dissolved in. Because a change in temperature leads to a change in the density of the liquid as well, temperature change is observed as a shift of the resonance line in the 129Xe NMR spectrum. This property can be utilized in the accurate determination of the sample temperature. Self-diffusion measurements of the gases provide additional information on a larger scale rather than just the immediate surroundings of atoms or molecules. Various liquid crystals were studied using xenon and SF6 as probes proving their applicability. It is often considered that the signal observed in NMR experiments is very weak and limits the full potential of the method. This is true especially with the samples in gaseous form. The Spin-Exchange Optical Pumping (SEOP) hyperpolarization method solves this problem in the case of xenon. A 129Xe NMR signal can be enhanced by a factor of 104–105 by SEOP and this opens access to techniques that are not otherwise possible. The remote detection technique, which separates the encoding and detection steps of the typical NMR experiment both temporally and spatially, is one of these techniques. The potential of the combination of SEOP and remote detection techniques was shown in studies of thermally modified Pinus Sylvestris.

Many devastating zoonotic viruses such as West Nile and Rift Valley fever viruses are endemic to South Africa, affecting livestock and ultimately, through their arthropod vectors, also infecting humans. One such zoonotic virus that is of interest is Shuni virus (SHUV). SHUV belongs to the viral genus Orthobunyavirus, family Peribunyaviridae., and order Bunyavirales. Discovered in arthropods and humans in Nigeria, it was soon identified as a possible cause for cases of neurological disease in horses within South Africa. Studies have shown South African veterinarians who had come into contact with such cases tested positive for antibodies against the virus. Therefore, SHUV is being further investigated as a potential cause of neurological disease within humans and there is a need to develop appropriate quick and effective diagnostic reagents to allow for surveillance of the virus. The main focus for this study was the development of diagnostic reagents centred around the nucleocapsid (N) protein of the SHUV. The N proteins of closely related members of the order Bunyavirales have shown to be highly abundant in infection and cause an immune response in the infected hosts thus making it the ideal target. Using available SHUV genome sequences and data, the N protein gene was designed and synthesised to be expressed in both Escherichia coli and plant expression systems. The expression of the N protein in E. coli, followed by subsequent washing with BugBuster, led to a final mass of 5.1 mg of the SHUV N protein from a 1000 ml culture. This led to a SHUV N yield of 5.1 µg/ml of culture and was measured to make up 69.5% of the total soluble protein. The immunisation of rabbits with this recombinantly expressed SHUV N allowed for the development of polyclonal antibodies which were successfully used in immunoblot studies to detect plant produced SHUV N protein. Plants are an effective and possibly cheaper alternative production system to bacterial, mammalian, or insect cell cultures and thus the N protein was transiently expressed in N. benthamiana plants using Agrobacterium tumefaciens-mediated infiltration. The recombinant protein produced underwent purification using nickel affinity chromatography. This led to yields of 2.248 mg of SHUV N protein from 35 plants which gave a yield of 9.9 mg/kg of raw plant material. This purified plant produced N protein acted as an antigen for diagnostic assays such as ELISA, which was used to screen known SHUV infected sera. This led to mixed results due to the limited sera samples available. However, as a proof of concept, it has shown great potential and thus opens the door to a possible inexpensive dual-use assay for use in the diagnoses of both animal and human SHUV infection.

Homocysteine is a natural occurring aminothiol. It is an intermediate product in the metabolism of methionine. Methionine is an essential amino acid required for protein synthesis. Metabolic irregularities disrupt homocysteine levels in plasma. Elevated homocysteine levels are directly linked to folate and cobalamin (vitamin B12) deficiencies, and are an independent risk factor for cardiovascular diseases. High homocysteine levels have also been associated with Alzheimer's, osteoporosis, renal failure, cancer, birth defects and pregnancy complications. The association of elevated homocysteine levels with cardiovascular disease and other diseases has generated great interest in the detection of homocysteine. An optical method for the detection of homocysteine has been developed using fluorescein mono- and dialdehydes. Selectivity for homocysteine was achieved based on the characteristic differences between 5- and 6-membered ring heterocyclic amines formed upon the reaction with fluorescein mono- and dialdehydes. 6-membered ring homocysteine-derived thiazinane-4-carboxylic acids were found to be more basic than 5-membered cysteine-derived thiazolidine-4-carboxylic acids. Fluorescence enhancement in response to homocysteine was thus attained by tuning pH and excitation wavelengths. Furthermore, the design and synthesis of a more sensitive fluorophore, fluorescein tri aldehyde has been accomplished based on the aforementioned findings to enable the detection of homocysteine at physiological levels. Calculations of Mulliken charges revealed that the formation of thiazinanes results in modulation of the electron density on the fluorophore leading to higher fluorescence.

The purpose of this research was to investigate the application of design processes to the development of novel self-use molecular diagnostic devices for sexually transmitted infections. The argument proposed in this thesis is that the application of design methods at the earliest research stages into miniaturised, low cost, molecular diagnostic technologies will accelerate and improve the process of translating proof of concept diagnostic technologies into usable devices. Concept development requirements and potential issues and barriers to development were identified through interviews with expert stakeholders. These requirements were further refined through a survey of a multidisciplinary diagnostic medical device research group. An action research method was applied to develop a proof of concept prototype to the preclinical trial stage. Through these research studies, a design process model was formulated for use in a research environment. The application of design methods to the proof of concept prototype described in the thesis have resulted in a preclinical trial prototype that exhibits the necessary features for development into a self-use molecular diagnostic device. Issues and barriers were identified and discussed, design guidelines for further development beyond preclinical trial were defined and a generalised design process model for self-use molecular diagnostic devices for sexually transmitted infections was proposed. This research highlights the need for design methods to be applied at the earliest possible stages of the development of novel molecular diagnostic devices.

The Foot-and-mouth disease virus (FMDV) affects cloven-hoofed animals and is endemic in most parts of Africa, South America and southern Asia. South Africa is considered a FMDV-free zone but the virus is maintained within the wildlife in the Kruger National Park (KNP), making mitigation of outbreaks a high priority. Diagnostic methods are usually costly due to the high production cost of the reagents used, meaning that regular monitoring and diagnosis of animals around the KNP for FMDV is expensive due to the large amounts of serum continuously being tested. I propose an alternative plant expression platform for the local production of more cost effective diagnostic reagents capable of distinguishing between infected and vaccinated animals (DIVA). I selected the non-structural 3ABC polyprotein of FMDV to express, as it is a suitable candidate as a coating antigen in a competitive enzyme linked immunosorbent assay (C-ELISA) for the detection of neutralizing antibodies in livestock sera. I also chose other variations of the full polyprotein (3AB, 3AB1 and 3B) for expression as they have previously been shown to be effective in FMDV diagnosis. I also selected a second reagent to be expressed: this was the CRAb-FM27 single chain variable fragment (scFv), which binds a 3B epitope on the 3ABC polyprotein and has previously shown to be effective as a competing antibody in a C-ELISA. The 3B antigen and the scFv were successfully expressed and purified from N. benthamiana, which to my knowledge is the first time either has been shown. The plant produced scFv successfully bound the 3B antigen in an I-ELISA. Separately, the plant produced 3B antigen could be used to successfully differentiate FMDV infected and vaccinated guinea pig serum in an I-ELISA. However, testing of these reagents in tandem within a C-ELISA to DIVA sera was inconclusive, and further research is required to optimise C-ELISA conditions.

Thesis (Honors paper)--Florida State University, 2009.
Advisor: Dr. Hank W. Bass, Florida State University, College of Arts and Science, Dept. of Biological Science. Includes bibliographical references.

Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill & Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis. Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined. The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications.
Patogena organismer smittas till värd organismen genom alla möjliga kontaktnätverk och skapar en mångfald olika sjukdomstillstånd. Dock är det fortfarande vanligt förekommande behandlingsbara infektiösa sjukdomar som orsakar den största hälsoförlusten, sett från ett globalt perspektiv. Bill och Melinda Gates Stiftelsen samarbetade med RAND kooperation för att forma “The Global Health Diagnostics Forum”. Deras mål var att etablera och analysera matematiska modeller för vilka effekter en ny diagnostisk metod utrustat för fältarbete skulle ha i utvecklingsländer. Resultaten var häpnadsveckande, med potentiellt miljoner av liv som skulle kunna räddas på en årlig basis. Den etablerade standarden för diagnostik av patogena bakterier har länge varit kultiveringsmedia baserad. Miljö specialiserade biologer har estimerat att mindre än 1 % av alla bakterie arter går att kultivera. Dock erbjuder genetiska analyser potentialen att kunna identifiera alla mikrober från alla de biologiska rikena. Nukleinsyrebaserade diagnostiska metoder har märkbart förbättrats över de senaste årtionden. Nya tekniker erbjuder utökad sensitivitet, selektivitet, sänkta kostnader och parallella analyser av patient prover. Dock är de flesta metoderna begränsade till standardiserade laboratoriemiljöer. För att konstruera en väl fungerande diagnostisk fältutrustning för användning i problem områden, behöver världsledande tekniker identifieras och kombineras. Fokuseringsområdet för denna doktorsavhandling har varit att utveckla och utföra nukleinsyrebaserade metoder för patogen diagnostik. Metoder och experimentella utförande applicerades på två distinkta system i) sökning av antibiotika resistens relaterade mutationer i den patogena bakterien Neisseria gonorrhoeae och ii) genotypning av det cancer orsakande Humana Papillomaviruset (HPV). Den första delen av studien inriktade sig mot utveckling av snabba, direkta och multiplexa Pyrosekvenserings baserade nukleinsyreanalyser. Med förbättrad provprepareringsmetodologi kunde vi detektera multipla HPV infektioner med högre sensitivitet än vad tidigare beskrivits med liknande metodologi. Den andra delen av studien fokuserades på multiplexa nukleinsyre amplifikationer med “Molecular Inversion Probe” tekniken med sista steg Pyrosekvenserings analys. “PathogenMip assay” erbjuder ett komplett detektionsprotokoll för alla kända patogena organismer. Vi introducerar även den nya “Connector Inversion Probe”, en “Padlock Probe” kapabel att genomföra kompletta gap fyllningar för multiplex nukleinsyre amplifiering.
QC 20100624

A Síndrome de Martin-Bell é uma forma hereditária de retardo mental determinada pela perda da expressão do gene FMR1 que está associado com a expansão da repetição dos tri-nucleotídeos citosina-guanina¬-guanina (CGG). Os indivíduos com um alto grau dessa expansão apresentam o silenciamento da expressão desse gene, com conseqüente alteração no desenvolvimento do sistema nervoso do embrião, causando danos neurológicos irreparáveis. A citogenética é uma metodologia clássica que contribui para o diagnóstico da síndrome em casos sem esclarecimentos, porém sua sensibilidade isoladamente em muitos casos é insuficiente para um diagnóstico positivo mesmo diante de características clínicas evidentes. Na busca de uma alternativa para suprir esta necessidade de definir exatamente as alterações encontradas em cada caso propôs-se a otimização de uma PCR de alta fidelidade no diagnóstico diferencial da Síndrome e simultaneamente comparar com os dados da citogenética clássica. Para tanto, foram coletadas amostras de sangue periférico de 102 pacientes e avaliou-se 100 a 150 metáfases para cada indivíduo pela citogenética clássica e para a PCR duplex. Os resultados demonstram pelo teste de Kruskal-Wallis que a PCR com a citogenética não apresentou diferenças significativas (p>0,05). Porém quando avaliadados pelo índice de Kappa sugere-se que os dois critéiros de diagnósticos devem ser utilizados simultaneamente com caracteristicas clínicas do paciente. A PCR mostrou-se rápida e com custo relativamente menor, sendo portanto, aplicável no auxílio ao aconselhamento genético dos indivíduos e suas famílias, bem como para um acompanhamento psico-pedagógico adequado.
The Martin-Bell Syndrome is a heredity mental retard form determined by the loss of FMR1 gene expression which is associated with the tri-nucleotides cytosine-guanine-guanine (CGG) repetition expansion. The individuals showing a high degree of this expansion present the silencing of this gene expression, with a consequent alteration of the embryo nervous system evolution, causing irreparable neurological damage The cytogenetics is a classical methodology that contributes for diagnosing the syndrome in cases without clarification, thus its sensitivity isolated in many cases is insufficient for a positive diagnosis even in front of evident clinical characteristics. On searching for an alternative to fulfill this need to define the found alterations in each case, an optimization of a high fidelity PCR to Syndrome diagnosis and simultaneously to compare with classical cytogenetics. Peripheral blood samples were collected from 102 patients for evaluating 100 to 150 metaphases evaluation by classical cytogenetics for each sample and to duplex PCR. The results showed by the Kruskal-Wallis test that the PCR with the cytogenetics have not presented significant differences (p>0,05). However, when evaluated by the Kappa index it was suggested that the two diagnosis criteria should be used simultaneously with patient s clinical characteristics. The PCR showed itself quick and with a relatively lower cost , and in this way, applicable in helping genetically counseling individuals and their families, as well as sending them to a more adequate psycho-pedagogical treatment.

With conventional methods of diagnosis, substantial overlaps are common due to an absence of the expected CSF findings clearly aligning to bacterial or viral infection. Reduced sensitivity is commonly observed chiefly due to empiric antibiotic treatment leading to bacterial culture negative results. This leads to costly hospital admissions and unnecessarily prolonged treatment for the unexplained aetiology, further compounded by routine viral diagnostics not being commonly implemented for meningitis diagnosis. We developed and validated in-house quantitative real-time (qPCR) multiplex assays to test for bacterial causes namely: Neisseria meningitidis (ctrA gene), Haemophilus influenzae (hpd gene) and Streptococcus pneumoniae (lytA gene) and viral causes namely: enterovirus (5' UTR), herpes simplex (UL30 gene) and mumps virus (Fusion protein gene). The qPCR assays were carried out on the Biorad CFX 96 real-time instrument. These validated assays were used to screen a cohort of suspected meningitis cases. The retrospective study included 300 paediatric patients aged from 60 days-12 years, over a 1-year period (November1, 2012 to November 30, 2013) with suspected meningitis presented to the outpatient departments of the Red Cross War Memorial Children‟s Hospital (RCCH) in Cape Town. Cerebrospinal fluid with abnormal chemistry and cell counts was selected and total nucleic acid was extracted with the QIAsymphony virus/bacterial DSP kit (QIAGEN, Valencia, CA). The median age of children was 19 months (IQR: 6-65 months). Among the screened 291 CSF samples, 7 (2.4%) cases Gram stain results were obtained along with relatively few cases with positive bacterial culture growth 4/291 (1.4%). Based on bacterial qPCR results, 8 (2.7%), 3 (1%) and 1 (0.3%) were positive for S. pneumoniae, N. meningitidis and H. influenzae respectively. A majority of cases were viral positives with enteroviruses being the dominant at 91/291 (31.3%) and mumps virus 3/291 (1%). No herpes simplex DNA was detected. The bacterial qPCR showed a sensitivity and specificity of 85.7% and 97.7% respectively when compared against a composite reference standard (CRS). We report an improvement with additional detected causes of bacterial meningitis and highlight the burden of the common viral causes. However, a large proportion of cases (63.6 %) have aetiology still unknown. PCR shows valuable in concluding viral aetiology in routine diagnosis.

Xanthene dyes are common fluorophores which have been widely used as molecular probes. The xanthene fluorophores can be used as highly selective optical sensors to detect disease biomarkers. A new fluorogenic dye containing an alpha, beta-unsaturated aldehyde moiety exhibits selective fluorescent signal enhancement in the presence of cysteine or peptides containing N-terminal cysteine residues. The mechanism is based on synergistic covalent and supramolecular interactions. A unique rhodamine boronic acid indicator is used in an optimized data collection protocol for wavelength- and time-dependent selectivity of various saccharides and nucleosides. One indicator is thereby capable of selectively distinguishing structurally related analytes in mixtures. Moreover, the rhodamine-based boronic acid responds linearly to increasing riboside concentrations in urine samples, potentially enabling the screening for inborn purine metabolism disorders.

Thesis (MScMedSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Important steps towards the global control of Tuberculosis include the improvement of diagnosis, the development of effective vaccines and the identification of correlates of protection/protective immunity to Mycobacterium tuberculosis. This study has of three objectives: 1. To validate the findings of a previous study that showed increased levels of IL-1β and decreased levels of IL-17 in children who are exposed to tuberculosis but remain uninfected compared to those who are exposed/infected and unexposed/uninfected. 2. To define the protective immunological phenotype in children with negative IGRA’s and TST following exposure to Mycobacterium tuberculosis. 3. To evaluate a number of cytokines in both serum and saliva samples of identified tuberculosis cases and controls for their diagnostic potential and to evaluate saliva as a possible new diagnostic sample type. The study designs were as follows: Objectives1, and 2: Children with documented tuberculosis exposure and with Mycobacterium tuberculosis infection as assessed through interferon gamma release assays, children with exposure but no infection and a control group with no exposure nor infection were investigated. These participants were selected according to their exposure and infection phenotypes from a larger TB household contact study that was conducted in communities in Cape Town. Whole blood was stimulated in QuantiFeron tubes overnight and ten cytokines were measured in antigen stimulated and unstimulated supernatants by Luminex multiplex Immunoassay. Differential production of cytokines in the three groups was evaluated. Objective 3. Saliva and serum samples were collected from thirty eight adults with suspected tuberculosis who were recruited from a community health centre in Cape Town, after which the levels of thirty three host markers were evaluated in the samples using the Luminex platform. The main findings of the studies included: 1. Increased levels of IL-1β and decreased levels of IL-17 in children who are tuberculosis exposed but remain uninfected compared to those who are exposed/infected and unexposed/uninfected could not be confirmed. 2. Immune responses other than IFN-γ are different in children with different exposure and infection phenotypes. Higher IL-23 and IL-33 levels in children with tuberculosis exposure without subsequent Mycobacterium tuberculosis infection compared to children with no exposure were shown. 3. In both the tuberculosis cases and controls, the levels of most markers were above the minimum detectable limit in both serum and saliva, but marker levels were not consistently higher in one sample type. The levels of fractalkine , IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF and IL-5 in saliva, and those of IL-6, IL-2, SAP and SAA in serum, were significantly higher in tuberculosis patients, in comparison to the levels obtained in those without active tuberculosis (p<0.05). The area under the ROC curve was ≥ 0.70 for most of these markers, thereby confirming their diagnostic potential for TB disease. The work presented in this thesis has identified markers that may grant an improved understanding on the mechanisms that are associated with protection against Mycobacterium tuberculosis in children. The preliminary results presented show that the identification of host markers in saliva is possible and the utility of saliva for the development of rapid immune-based tests for active tuberculosis is promising.
AFRIKAANSE OPSOMMING: Noemenswaardige vooruitgang in die globale beheer van Tuberkulose is onderworpe aan verbeterde diagnose, die ontwikkeling van doeltreffende vaksienes en die identifikasie van aanwysers van immuniteit teen Mycobacterium tuberculosis. Die doel van hierdie studie is: 1. Om die bevindinge van ‘n vorige studie te bevestig, waar verhoogde vlakke van IL-1β en verlaagde vlakke van IL-17 waargeneem is in kinders wat aan tuberkulose blootgestel is, maar nie geïnfekteer is nie. Hierdie bevindinge was in vergelyking met geïnfekteerde en nie-blootgestelde kinders. 2. Om ‘n beskermende immunologiese fenotipe te definieer in kinders met negatiewe IGRA’s en TST, na blootstelling aan Mycobacterium tuberculosis. 3. Om sekere sitokines, in beide serum en speeksel monsters van tuberkulose gevalle en kontroles, te evalueer as potensiële diagnosemiddels, asook die moontlikheid dat speeksel kan dien as ‘n nuwe diagnostiese monstertipe. Die studieraamwerk was as volg: Doel 1 &2:Die volgende groepe was onder meer ondersoek – Kinders blootgestel aan tuberkulose en wat gevolglik geïnfekteer is, soos vasgestel deur interferon gamma vrystellingstoetse; kinders wat wel blootgestel is maar nie geïnfekteer is nie en ‘n kontrolegroep wat geen blootstelling aan Mycobacterium tuberculosis gehad het nie. Hierdie individue is geselekteer volgens hul blootstellingsprofiel en infeksiefenotipes, uit ‘n groter blootstellingstudie op Kaapse huishoudings. Heelbloed is oornag gestimuleer en tien sitokiene is gemeet in antigeen-gestimuleerde en ongestimuleerde supernatante, deur middel van Luminex multipleks Immunotoetse. Differensiële produksie van sitokienes in hierdie groepe is gevolglik geëvalueer Doel 3: Speeksel en serummonsters van 38 volwassenes met vermeende tuberkulose, is versamel en die vlakke van drie en dertig gasheermerkers is gemeet deur middel van die Luminex platvorm. Die hoof bevindinge van hierdie studie sluit in: 1.Vehoogde vlakke van IL-1β en verlaagde vlakke van IL-17 kon nie bevestig word in die verskeie kindergroepe (Sien doel 1) nie. 2. Die immuunrespons, uitsluitend die IFN- γ respons, is veskillend in kinders met uiteenlopende blootstelling en infeksiefenotipes. Hoër vlakke van IL-23 en IL-33 is gevind in kinders wat blootgestel is aan tuberkulose, maar nie geïnfekteer is nie, in teenstelling met nie-blootgestelde kinders.. 3. In beide die pasiënte en kontroles was die meeste sitokienvlakke hoër as die minimum meetbare limiet in beide speeksel en serummonsters, hoewel merkervlakke nie konstant hoër was in enige van die twee monstertipes nie. Die vlakke van fractalkine, IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF en IL-5 in speeksel en IL-6, IL-2, SAP en SAA in serum, was merkbaar hoër in tuberkulosepasiënte, in vergelyking met vasgestelde vlakke in individue sonder aktiewe tuberkulose. (p<0.05). Die oppervlak onder die ROC kurwe was ≥ 0.70 vir die meerderheid van die merkers. Dit is ‘n sterk aanduiding dat hierdie merkers potensiaal het as diagnostiese merkers vir tuberkulose. Hierdie navorsing het merkers geïdentifiseer wat die begrip van die megansime waarmee beskerming teen Mycobacterium tuberculosis gebied word in kinders, verbreed. Hierdie voorlopige resultate dui aan dat die identifikasie van gasheermerkers in speeksel moontlik is en dat speeksel moontlik kan dien as ‘n proefkonyn vir die ontwikkeling van immuungebaseerde sneltoetse vir die diagnose van aktiewe tuberkulose.
The EDCTP through the African European Tuberculosis Consortium (AE-TBC, grant number IP_2009_32040)
Trials of Excellence in Southern Africa (TESA, project code CG_cb_07_41700)

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that has devastated the global swine industry since the mid 1980s. Although modified live vaccines (MLVs) are typically used for the prevention of clinical disease, they are not always fully effective. Additionally, acute PRRS outbreaks, characterized by more severe clinical signs, have appeared in herds that were previously vaccinated. In this dissertation, we further analyzed the pathogenesis of PRRSV through genetic characterization, assay development, and quasispecies evaluation using the PRRSV ORF5 gene while also attempting to develop an improved PRRS vaccine. To explore the possible mechanism for the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. Sequence and phylogenetic analyses revealed that seven of the acute PRRS virus (PRRSV) isolates were related to other N. American PRRSV isolates while one isolate, 98-37120-2, was very closely related to and may have been derived from the MLV, RespPRRS. We also developed a heteroduplex mobility assay (HMA) for quickly identifying PRRSV field isolates with significant nucleotide sequence identities (â d98%) with the MLVs based on the amplification, denaturation, and reannealing of the ORF5 gene of the field isolates with those of MLV reference strains. All of the field isolates that were highly related to RespPRRS (â T2% nucleotide sequence divergence) were identified by the HMA to form homoduplexes with the reference RespPRRS MLV. We also developed a unique strategy for infecting pigs with PRRSV, known as in vivo transfection, by bypassing the traditional in vitro cell culture step required for in vivo studies. We demonstrated that inoculation of RNA transcripts of a PRRSV infectious cDNA clone directly into the lymph nodes and tonsils of pigs produces active PRRSV infection. Using this method, we also examined the quasispecies populations of PRRSV. Finally, we evaluated the ability of Salmonella choleraesuis to express the PRRSV GP5, and tested its immunogenicity in mice. Based on our data, there was no indication of Salmonella replication in the mice or any evidence of antibody production against S. choleraesuis or PRRSV GP5.
Ph. D.

L’imagerie moléculaire optique joue maintenant un rôle essentiel dans le diagnostic pré-clinique et le développement de médicaments. En effet, c’est un outil précieux dans la détection et le suivi de cellules vivantes que ce soit en utilisant de simples agents de marquage ou des sondes plus développées, dites « intelligentes » et activées uniquement par une interaction spécifique avec le bio-analyte ciblé. Ce travail de thèse a consisté à développer des outils synthétiques innovants afin d’optimiser les paramètres physico-chimiques et les propriétés optiques des sondes luminescentes. Ceci dans le but de répondre à la problématique complexe de l’imagerie dans le contexte in vivo. Nous avons notamment travaillé sur des aspects de pro-fluorescence et de chémiluminescence. De nouveaux pro-fluorophores à phénol basés sur une architecture originale de type bis-coumarinique ont été développés. De plus, nous avons mis en place une méthode d’hydrosolubilisation généralisable aux fluorophores à phénol de type coumarine et xanthène. Nos recherches en chémiluminescence ont permis la synthèse de nouveaux chémiluminophores couplés à des fluorophores organiques afin d‘augmenter l’efficacité d’émission de chémiluminescence dans le rouge. Enfin, nos travaux ont permis de mettre en place les premières « cassettes » chémiluminescentes basées sur une architecture de type 1,2-dioxétane
Optical molecular imaging is now playing a pivotal role both in pre-clinical diagnosis and drug development. Indeed, this is a valuable tool for the real time detection and monitoring of living cells either through the use of structurally simple labels or more recently by means of sophisticated fluorescent probes, called “smart” probes and only activatable upon specific interaction with the targeted bio-analyte. The aim of this PhD work was the design of new synthetic tools aimed at optimizing physico-chemical and optical properties of fluorescent probes intended for challenging in vivo imaging applications. We have focused on the pro-fluorescence and chemiluminescence approaches. New phenol-based pro-fluorophores have been developed by using an original bis-coumarinic scaffold. In the context of the chemistry of fluorophores, we have also investigated a general method for the water-solubilisation of phenol-based fluorophore belonging to the coumarin and xanthene families. Our research in chemiluminescence has led the synthesis of new chemiluminophores covalently linked to fluorescent organic dyes aimed at increasing the emission efficiency in the red region of such chemiluminophores. Thus, the first chemiluminescent “energy transfer cassettes” based on a 1,2-dioxetane scaffold have been obtained

Yeung Kwun Yan.
Thesis submitted in: December 2000.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 106-122).
Abstracts in English and Chinese.
Acknowledgements --- p.iv
Table of Contents --- p.v
List of Tables --- p.viii
List of Figures --- p.ix
Abbreviations --- p.x
Conference Presentations --- p.xii
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Retinitis Pigmentosa (RP) --- p.1
Chapter 1.1.1 --- Molecular genetics --- p.1
Chapter 1.1.2 --- Clinical features --- p.2
Chapter 1.1.3 --- Clinical classifications of RP --- p.3
Chapter 1.2 --- Molecular Biology of Rhodopsin --- p.4
Chapter 1.2.1 --- Anatomy and functions of human retina --- p.4
Chapter 1.2.2 --- Physiology of rhodopsin --- p.5
Chapter 1.2.3 --- Rhodopsin cycle and the visual transduction cascade --- p.7
Chapter 1.2.4 --- The human rhodopsin gene (RHO) --- p.8
Chapter 1.2.5 --- RHO mutations --- p.8
Chapter 1.2.6 --- Frequencies & phenotypes ofmutations --- p.10
Chapter 1.2.7 --- Findings of in vitro experiments --- p.11
Chapter 1.2.8 --- Findings in animal models --- p.12
Chapter 1.2.9 --- Findings in human --- p.14
Chapter 1.3 --- Molecular Biology of RP1 --- p.15
Chapter 1.3.1 --- RP1 gene in animals --- p.16
Chapter 1.3.2 --- Mutations in RP1 --- p.16
Chapter 1.3.3 --- Phenotypes & frequencies of RP1mutations --- p.17
Chapter 1.4 --- Mutation Pattern of RHO & RP1 in Chinese --- p.18
Chapter 1.5 --- Methods for Detecting Mutations in RHO and RP1 --- p.18
Chapter 1.6 --- Management of RP --- p.20
Chapter Chapter 2 --- Study Objectives --- p.31
Chapter Chapter 3 --- Methodology --- p.32
Chapter 3.1 --- Study Subjects --- p.32
Chapter 3.2 --- Clinical Data Sheet --- p.32
Chapter 3.3 --- "Chemicals, Reagents, and Kits" --- p.35
Chapter 3.4 --- Solutions and Buffers --- p.36
Chapter 3.5 --- Enzymes --- p.37
Chapter 3.6 --- Equipment --- p.37
Chapter 3.7 --- Software --- p.38
Chapter 3.8 --- "Oligonucleotide Primers for PCR, CSGE and Sequencing" --- p.38
Chapter 3.9 --- DNA Extraction --- p.38
Chapter 3.9.1 --- DNA extraction from blood samples --- p.39
Chapter 3.9.2 --- DNA extraction from buccal swab --- p.39
Chapter 3.9.3 --- DNA quantitation --- p.39
Chapter 3.10 --- Polymerase Chain Reaction (PCR) --- p.40
Chapter 3.10.1 --- Amplification of RHO --- p.40
Chapter 3.10.2 --- Amplification of RP1 --- p.40
Chapter 3.11 --- Gel Electrophoresis --- p.40
Chapter 3.11.1 --- Agarose gel electrophoresis --- p.41
Chapter 3.11.2 --- Conformation sensitive gel electrophoresis (CSGE) --- p.41
Chapter 3.11.3 --- DNA sequencing --- p.42
Chapter 3.12 --- Statistical Methods --- p.43
Chapter Chapter 4 --- Results --- p.51
Chapter 4.1 --- Study Subjects --- p.51
Chapter 4.1.1 --- RP index patients --- p.51
Chapter 4.1.2 --- Family members of index patients --- p.51
Chapter 4.1.3 --- Controls --- p.51
Chapter 4.2 --- Genetic subtypes of RP in our study --- p.52
Chapter 4.3 --- PCR --- p.52
Chapter 4.4 --- Conformation Sensitive Gel Electrophresis (CSGE) --- p.53
Chapter 4.5 --- Direct DNA Sequencing --- p.53
Chapter 4.5.1 --- Sequence alterations in RHO --- p.54
Chapter 4.5.2 --- Sequence alterations in RP1 --- p.56
Chapter 4.6 --- Family Studies --- p.60
Chapter Chapter 5 --- Discussion --- p.77
Chapter 5.1 --- The Expected Frequencies of RHO & RP1 Mutationsin Chinese RP Patients --- p.82
Chapter 5.2 --- The Mutation Screening Technique in this Study --- p.84
Chapter 5.3 --- Mutations and Sequence Alterations Identified in RHO --- p.86
Chapter 5.3.1 --- Novel mutation: 521 ldelC --- p.86
Chapter 5.3.2 --- Reported mutation: Pro347Leu --- p.90
Chapter 5.3.3 --- Novel nonpathogenic missense change: Ala299Ser --- p.92
Chapter 5.3.4 --- Novel silent sequence alterations --- p.93
Chapter 5.3.5 --- Other polymorphisms in RHO --- p.93
Chapter 5.4 --- Mutation and Sequence Alterations Detected in RP1 --- p.94
Chapter 5.4.1 --- Mutation found in Chinese: Arg677ter --- p.95
Chapter 5.4.2 --- Novel nonsense sequence alteration: Arg l933ter --- p.96
Chapter 5.4.3 --- Novel missense and non-coding changes in RP1 --- p.97
Chapter 5.4.4 --- Reported polymorphisms --- p.98
Chapter 5.5 --- Possible Functions of RP1 --- p.99
Chapter Chapter 6 --- Conclusion --- p.105
Chapter Chapter 7 --- References --- p.106

博士
國立中興大學
食品科學系
85
AbstractTwo major parts are included in this thesis. The first one is aimed for the development of 16S rDNA targeted PCR primers which could be used for the specific detection of Serratia marcescens and S. rubidaea. The second part of this thesis, on the other hand, is aimed for the subtyping of Salmonella typhimurium and Sal. typhi strains collected in Taiwan and obtained from USA. Those subtyping data may be useful for tracing the contamination sources, the transmission path and clonal relationship for these bacterial strains. By comparing the 16S rRNA sequence of some enterobacteria, primers SM2/SMR were designed which could be used for the detection of 46 strains of S. marcescens and 3 strains of S. rubidaea although S. rubidaea SER43 gave the negative result. After 16S rRNA V1 region of S. marcescens SER10 and S. rubidaea SER40 were PCR amplified and sequenced. Based on such sequences, SM3/SM4 primers were designed for the detection of S. maecescens.When primers SM3/SM4 were designed and used for the detection of S. marcescens, all the 46 S. marcescens strains generated positive result and all the non-S. marcescens strains tested would not give the false positive result. The detection limits for SM3/SM4 primers was Nx100 CFU for S. marcescens cultures and Nx100 ~ 101 target cells per 100 ml of blood sample.After the sequence analysis for 16S rRNA gene of S. rubidaea CCRC 13990, it was found that there were two different DNA sequences, ie, 16S1 and 16S2 sequences, in the V6 region. Four strains of S. rubidaea posses 16S1 and 16S2 sequences while S. marcescens CCRC 10948 posses only the 16S2 sequence. S. rubidaea SER43 are different from other 3 S. rubidaea strains when their sequences in V1 and V3 regions are compared. Thus, it may be necessary to reconfirm the SER43 strain. PCR primers SR1/SM4 allowed the detection of S. rubidaea strains SER40, 41 and 42 with no false positive reaction from strains other than S. rubidaea. On the subtyping work of Salmonella strains, we found that 6 PCR ribotyping patterns could be found for the 153 S. typhimurium strains. On the other hand, when primer set of OPF-13 were used for RAPD analysis, 40 RAPD patterns could be found for these 153 strains of S. typhimurium. PFGE analysis of the XbaI digested chromosomal DNAs for these S. typhimurium obtained in Taiwan and USA showed that 81 PFGE patterns could be observed. Of these PFGE patterns, 19 patterns(f1 to f19) are found for the 45 Taiwan iIn conclusion, our results may imply that in molecular typing of bacterial strains, PFGE technique is a rather reliable and accurate method while RAPD and PCR ribotyping methods, when in combination with the PFGE method, may be helpful in tracing the clonal relationship for pathogenic bacteria strains.

Indiana University-Purdue University Indianapolis (IUPUI)
Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative form of GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). We also demonstrate that GCN5b interacts with AP2-domain proteins, which are plant-like transcription factors in Apicomplexa. The interactions between GCN5b, AP2IX-7, and AP2X-8 were confirmed by reciprocal co-immunoprecipitation and revealed a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.