Understanding the biological basis of behavior is crucial for gaining insights into human nature, treating behavioral disorders and improving overall well-being. Efforts to understand the biological basis of behavior have largely emphasized the role of neurons. However, examples across life show that behavior can occur in lieu of or in cell types outside of neurons. This thesis presents work exploring the mechanisms underlying multiple behaviors using the model system Drosophila melanogaster. Chapter 2 provides evidence for the influence of glia on courtship, aggression, and sleep. Chapter 3 characterizes a Drosophila model of autism spectrum disorder, finding that genetic neuronal manipulation of a single gene can cause pathologies beyond the nervous system. Chapter 4 investigates the connection between sleep behavior and oxidative stress response, demonstrating metabolism as a probable mediator of this relationship. Altogether, this work supports a wider definition for the biological basis of behavior.

Diffuse Midline Glioma (DMG) are universally fatal, primarily pediatric malignancies affecting the midline structures (i.e., pons, thalamus, and spinal cord) of the central nervous system. Despite decades of clinical trials, no drugs have emerged as effective against this disease, and treatment remains limited to palliative radiation therapy. Primary treatment challenges include: A) Well-stablished, yet non-actionable, genetic alterations; B) significant intratumoral heterogeneity, and C) blood-brain barrier (BBB) drug permeability. Here, we address the former two challenges by leveraging network-based methodologies to dissect the heterogeneity of DMG tumors and to discover Master Regulators (MR) proteins representing pharmacologically accessible, mechanistic determinants of molecularly distinct DMG cell states. We reverse engineered the first DMG gene regulatory network from 122 publicly available DMG RNA-seq profiles with ARACNe and inferred sample-specific MR protein activity with VIPER based on the differential expression of their targets. Nine of the top 25 most active MRs (i.e., FOXM1, CENPF, TOP2A, ASF1B, E2F2, TIMELESS, MYBL2, CENPK, TRIP13) comprise a well-characterized MR block (MRB2), frequently activated across aggressive tumors, and found to be enriched in DMG patient MR signatures (Fisher’s Exact Test p = 3.96x10-16). A pooled CRISPR/Cas9-mediated knockout (KO) screen across three DMG patient cell lines targeting 1,433 genes identified a set of 73 essential genes that were enriched in the MR signature of 80% of patient samples (GSEA p = 0.000034). FOXM1 emerged as a highly essential MR, significantly activated across virtually all patients. We then generated drug-induced differential protein activity from RNA-seq profiles following perturbation with 372 oncology drugs in two DMG cell lines that together recapitulate DMG patient MR and used this to identify drugs that invert patient MR activity profiles using the NYS/CA Department of Health approved OncoTreat algorithm OncoTreat predicted sensitivity to HDAC, MEK, CDK, PI3K, and tyrosine kinase inhibitors in subsets of patients, overlapping with published DMG drug screens. Importantly, 80% of OncoTreat-predicted drugs (p < 10-5) from three DMG patient tumor biopsies showed in vitro sensitivity in cultured tumor cells from the respective patients, with overall 68% accuracy among 223 drugs evaluated by both OncoTreat and in vitro drug screen (Fisher’s Exact Test p = 0.0449). Given known resistance in DMG to single-agent therapy, we further interrogated single-cell DMG regulatory networks generated by ARACNe with gene expression signatures from 3,039 tumor cells previously published across six patients using VIPER to infer single-cell regulatory protein activity. Unsupervised clustering of cells by protein activity defined 7 patient-independent cell states with distinct MR profiles reflecting known glial lineage markers (OPC-like-S1, OPC-like-S2, OC-like-S1, OC-like-S2, Cycling, AC-like, and AC/OPC-like). We identified drugs that invert the MR activity profiles of the individual cell states by using OncoTarget (inhibitors of individual MRs) or OncoTreat using the drug-induced differential protein activity we previously generated. Predicted drugs were distinct across the previously defined cell states with bulk RNA-seq recapitulating predictions seen in the more prevalent OPC-like stated, but failing to recapitulate the MRs and drug predictions for the smaller AC-like stated. We selected five drugs targeting the OPC/cycling-like cells (Trametinib, Dinaciclib, Avapritinib, Mocetinostat, and Etoposide), and four drugs targeting the AC-like cells (Ruxolitinib, Venetoclax, Napabucasin, Larotrectinib) for further validation as these states comprised most tumor cells across patients. We then generated single-cell RNA-seq for 95,687 cells after 5 days of treatment with either vehicle control (n = 4) or candidate drug (n = 2-3/drug) in subcutaneous SU-DIPG-XVII patient cell line-derived mouse models. We show this model recapitulates DMG cell states seen in patients, and confirm reduction in tumor growth and significant depletion of either OPC/cycling-like cells or AC-like cells in line with our drug predictions for 8/9 candidate drugs (Chi-square p<0.01). We further treated a syngeneic (DIPG4423) orthotopic DMG model with each drug and demonstrate significant differences in survival with Avapritinib, Dinaciclib, and Trametinib. Notably, the combination of drugs targeting OPC/cycling-like and AC-like cells (i.e. Trametinib+Ruxolitinib, Dinaciclib+Ruxolitinib, Avapritinib+Venetoclax, etc.) showed significantly lower tumor volumes after 2 weeks of treatment as compared to vehicles or each drug alone, and significant survival differences for some of the combinations. This work provides a precision medicine platform to nominate much-needed novel drug combinations addressing DMG tumor heterogeneity for further study to improve outcomes in this devastating disease.

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The gram-negative bacterial pathogen Brucella abortus is a zoonotic pathogen causing brucellosis in a variety of animal species including humans. The loss of the O-side chain in the lipopolysaccharide of the outer membrane decreases Brucella virulence. To understand the genetics of O-side chain biosynthesis and its relationship to virulence, studies were initiated to characterize specific O-side chain mutants. B. abortus rough mutant strain RA2 was derived by transposon (Tn5) mutagenesis of smooth B. abortus 2308. The chromosomal region of strain RA2 with the Tn5 and flanking chromosomal region was cloned into the sequencing vector pGEM-3Z to create a suicide plasmid pNW-2. The plasmid pNW-2, or a derivative of it (pNW-3), in which Tn5 was replaced with a Kank gene, were electroporated into wild type smooth B. abortus 2308 in order to assess the phenotypic conversion from smooth to rough. The electroporation parameters such as cell growth stage, pulse field strength and pulse length were optimized. It was determined that using late log phase cells (approximately 70-77 Klett units), 10 ms and 13 KV/cm were the best conditions for achieving transformation by pNW-2 or pNW3. Kanamycin resistant and ampicillin sensitive Brucella were screened for double reciprocal crossovers between the suicide plasmids (pNW-2 and pNW-3) and Brucella chromosomal DNA. The recombinants were checked for their O-side chain by crystal violet uptake and immunoblotting with monoclonal antibody specific for the O-side chain. The locations of Tn5 and the flanking region in the genome of these recombinants were characterized by Southern blot using either a Tn5 probe or a flanking region probe. An analysis of KanR colonies showed that none of the recombinants were rough. The B. abortus DNA in pNW-2 was sequenced and compared with other genes. No Significant homology was found between the Brucella DNA in pNW-2 and gene sequences in the gene bank. Analysis of the recombinants suggests no linkage between the Tn5 element in strain RA2 and the rough phenotype. / Master of Science

Because the many physicians and students of heredity have not been closely associated in finding that the causes of cardiovascular diseases might lie, in a large measure, to an inherited predisposition, this study has been undertaken; first, to contribute to several more case studies of cardiovascular diseases to the knowledge of heredity; second, to find if there is a possible successive occurrence of cardiovascular diseases in generation after generation; and third, to determine a possible mode of transmission from parents to offspring by an analysis of nine genealogies.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: With knowledge of the human genome increasing constantly we are continually faced with new and potentially groundbreaking methods for managing, treating and/or identifying diseases and predisposition to diseases and conditions at a genetic level. The human cytochrome P450 (CYP) super-family of genes code for enzymes that can participate in metabolism of drugs and foreign chemicals and in steroid synthesis and metabolism. Mutations in these genes may contribute to clinically relevant diseases. In this study, the effects of mutations within four CYP genes were evaluated in two South African disease groups - variegate porphyria and breast cancer. Variegate porphyria (VP) has an unusually high incidence in South Africa due to the R59W founder mutation in the protoporphyrinogen oxidase (PPOX) gene that causes a disruption in the haem biosynthetic pathway. VP presents with variable clinical symptoms and has a relatively low penetrance. It is expected that environmental factors and modifier genes play a role in the clinical expression of VP. CYP genes are implicated as candidate modifier genes for the expression of VP due to the function they have in metabolising many drugs contraindicated in porphyria patients, and the necessity of haem binding to the apoprotein to produce a functional CYP enzyme. This is the first study to investigate CYPs as possible modifier genes for VP clinical expression. Six CYP polymorphisms (CYPIAlml, CYPIAlm2, CYPIA2 - 734 C>A, CYPIBI 8372 A>C, CYP2D6*3, CYP2D6*4), associated with four CYP loci, were genotyped in a VP population and a suitable control population. The results observed are suggestive of CYPIAlml and CYPIBI playing a role as modifiers for the clinical expression of VP as they were significantly associated (P<O.05) with the presence ofVP related symptoms. Breast cancer is one of the leading causes of death in women due to cancer. It is believed that breast cancer may be the result of co-participation between common DNA alterations and environmental exposures. Polymorphisms in enzymes involved with the metabolism of environmental exposures, such as the cytochrome P450 enzymes, are therefore expected to play an aetiological role in breast cancer. Two groups of South African breast cancer patients (Caucasian and of mixed ancestry) and population-matched controls were genotyped for four CYP polymorphisms (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A and CYPIBI 8372 A>C). This represents the first investigation of the potential role of CYPs as breast cancer risk modifiers in the two South African populations. Significant differences were observed (P<O.003) in genotype and allele frequencies between the breast cancer patients and controls for the CYPIBI 8372 A>C polymorphism in the population of mixed ancestry. Vast differences in allele frequencies were also observed between the two groups of breast cancer populations. These results emphasize the importance of population-based risk assessment when genetic testing and counselling for complex disease susceptibility is offered. The results of this study provide the first evidence suggesting a role for CYPs in modifying the clinical expression of VP and in acting as risk factors for developing breast cancer in a South African population. / AFRIKAANSE OPSOMMING: Met die konstante toename van kennis oor die mensgenoom kom ons voortdurend te staan voor nuwe metodes vir die beheer, behandeling en/of identifikasie van siektes en vatbaarheid vir siektes op 'n genetiese vlak. Die mens sitochroom P450 geensuperfamilie kodeer vir ensieme betrokke in die metabolisme van medisyne en ander chemiese stowwe en steroïed-sintese en -metabolisme. Mutasies in hierdie gene kan 'n bydrae lewer tot kliniese relevante siektes. In hierdie studie is die effek van mutasies in vier sitochroom gene bestudeer in twee Suid-Afrikaanse siekte groepe, variegate porfirie en borskanker. Variegate porfirie (VP) het 'n besonderse hoë frekwensie in Suid-Afrika as gevolg van die R59W stigter-mutasie in die protoporfirinogeen oksidase (PPOX) geen. Hierdie mutasie lei tot 'n versteuring in die heem biosintese padweg. VP presenteer met variërende kliniese simptome en het 'n betreklike lae penetrasie. Daar word vermoed dat omgewingsfaktore en kandidaat modifiserende gene 'n rol speel in die kliniese beeld van VP. Sitochroom P450 gene is geïdentifiseer as kandidaat modifiserende gene as gevolg van hulle rol in die metabolisme van verbode medikasie vir porfirie pasiënte, asook die binding van heem aan die apoproteïen wat noodsaaklik is vir die produksie van funksionele sitochroom P450 ensiem. Hierdie is die eerste studie wat sitochroom P450 gene as moontlike modifiserende gene vir die kliniese uitdrukking van VP ondersoek. Ses sitochroom P450 polimorfismes (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A, CYPIBI 8372 A>C, CYP2D6*3, CYP2D6*4) is ondersoek in beide 'n VP populasie en 'n geskikte kontrole populasie. Die resultate suggereer 'n rol vir CYPIAlml en CYPIBI in die modifisering van die kliniese uitdrukking van VP aangesien hulle betekenisvolle assosiasie (P<O.05) met die voorkoms van VP-verwante simptome getoon het. Borskanker IS een van die vernaamste oorsake van kankersterftes onder vroue. Borskanker IS waarskynlik die resultaat van interaksie tussen algemene DNSveranderinge en omgewings-blootstelling. Daar word dus verwag dat polimorfismes in ensieme betrokke by die metabolisme van omgewingselemente, soos byvoorbeeld die sitochroom P450 ensieme, 'n etiolgiese rol in borskanker sal speel. Die genotipes van twee groepe Suid-Afrikaanse borskanker lyers (Kaukasiërs en van gemengde herkoms) en populasie-passende kontroles is bepaal vir vier sitochroom P450 polimorfismes (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A, CYPIBI 8372 A>C). Hierdie studie verteenwordig die eerste ondersoek na die potensiële rol van sitochroom P450s as risiko-modifiserende faktore vir borskanker in die twee populasies. Betekenisvolle verskille (P<O.003) in genotipe en allele frekwensies is waargeneem tussen die borskanker lyers en kontroles vir die CYPIBI 8372 A>C polimorfisme in die gemengde herkoms populasie. Beduidende verskille in alleel frekwensies is ook waargeneem tussen die twee borskanker populasies. Hierdie resultate beklemtoon die belangrikheid van populasie gebaseerde risiko-beraming wanneer genetiese toetse en voorligting vir komplekse siekte-vatbaarheid aangebied word. Die resultate van hierdie studie bied die eerste getuienis dat sitochroom P450s 'n rol kan speel in die modifisering van die kliniese beeld van VP en ook kan optree as as risiko faktore vir die ontwikkeling van borskanker in 'n Suid-Afrikaanse populasie.