A DNA probe was developed for the identification of the periodontal pathogen, Actinobacillus actinomycetemcomitans. Chromosomal DNA was extracted from A. actinomycetemcomitans, digested with a restriction enzyme, Sau3A, ligated to plasmid DNA (pUC18) and transformed into JM109 cells to give a partial A. actinomycetemcomitans library. The library was screened using Southern blot analysis. Out of the nine inserts tested, one was found to be species specific as it did not cross-hybridise to Haemophilus aphrophilus, a closely related organism which occurs in the normal oral microflora, nor did it cross-hybridise with 7 species of Bacteroides tested. A level of detection of 104 cells or 50ng of A. actinomycetemcomitans was obtained. The probe has a length of 779bp and out of 30 restriction enzymes tested, only SspI was found to have a restriction site in the insert. The probe was tested on clinical specimens obtained from five different periodontitis patient groups and was shown to correlate with culture results in eighteen out of twenty-two cases in detecting A. actinomycetemcomitans.

Indiana University-Purdue University Indianapolis (IUPUI) / The problem behavior theory suggests that the engagement in one problematic behavior increases the likelihood of engagement in another problematic behavior. Previous research has found among youth an increasing probability of co-occurring dieting and substance use behavior, particularly among girls. However, to date findings are inconclusive on the temporal ordering of these behaviors. Further, limited research has been conducted to explore whether the temporal ordering of the two behaviors exist similarly between White and Black youth. The present study will use a cross-lagged panel design across one year to examine the temporal ordering between dieting behavior and substance use among a sample of 2,016 adolescent females (grade mean=7; 77.2% White; 22.8% Black). We hypothesized that a bidirectional relationship would be observed between the two behaviors. However, given no published studies on this relationship by race, no a priori hypotheses were made for this second aim. Result showed that within the full sample dieting behavior significantly predicted substance use one year later, but the inverse relationship was not found. Additionally, this effect was replicated in the White sample but null effects in both directions was found among Black youth. These findings provide support for a temporal relationship between dieting behavior and substance use, such that the former predicts risk for the latter. Moreover, although there is evidence of race differences in the risk pathway, further research is needed to confirm this effect. Future studies are also needed to determine whether this observed temporal relationship is present among adolescent females of other racial/ethnic groups, as well as if the relationship varies as a function of other demographic variables, such as age (e.g., early, mid, or late-adolescence).

No description available.

No description available.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). Neuropathological studies in both human MS and the experimental autoimmune encephalomyelitis (EAE) animal model have shown that endothelial cell (EC) inflammation, associated with focal breakdown of the blood-brain barrier (BBB) and neo-angiogenesis, is prevalent in demyelinating plaques. Neo-angiogenesis and BBB damage contribute to leakage of serum components, infiltration of immune cells into the CNS, neuroinflammation, axonal demyelination, neuronal dysfunction, and disease progression. In Chapter 1, I introduce MS and its pathological hallmarks related to immune and vascular dysfunctions, the clinical course of MS progression, genetic and environmental influences, current treatments, and animal models. Next, I elaborate upon the pathways and processes involved in the development of a functioning CNS vascular system and the BBB. Finally, I discuss what is currently known about the contribution and the underlying mechanisms of neo-angiogenesis in MS and other diseases. While an increase in vessel density has been documented for both MS and EAE lesions, the origin and pathways that drive formation of new, but leaky, blood vessels in EAE are poorly understood. In Chapter 3, I address these questions by performing single-cell RNA-sequencing (scRNA-seq) of 45309 ECs isolated from the spinal cord of control, acute and chronic MOG35-55 EAE mice. Based on expression patterns of blood vessel subtype-specific markers, I identified 23 distinct EC clusters with arterial, capillary, venule, and vein identities in either control or disease states. I performed differential gene expression and gene set enrichment analyses comparing control and disease EC clusters for each vascular subtype to identify which vessels exhibited gene expression profiles indicative of neo-angiogenesis in EAE. I found that molecular signatures of neo-angiogenesis are upregulated specifically in venous ECs during acute and, to a lesser extent, chronic EAE. Consistent with these data, EC proliferation is upregulated in veins in the EAE spinal cord. RNA fluorescent in situ hybridization and immunofluorescence staining confirmed increased expression of key angiogenic markers Egfl7, Ecm1, Serpine1 and Emcn, and the tip cell marker Mcam, with a corresponding increase in vein density, in demyelinating white matter lesions of EAE spinal cords relative to controls. I also assessed changes in expression of some of these markers in human MS tissue and discovered upregulated expression of EGFL7 in cortical white matter lesions of MS patients, concomitant with increased vascular density. In Chapter 4, I examine the signaling pathways that may trigger pathogenic angiogenesis in EAE. I discovered that, in contrast to developmental angiogenesis, VEGF-A and TGF-β signaling may act as the driver of neo-angiogenesis in EAE. To test this hypothesis, I used a humanized VEGF-A blocking antibody, bevacizumab, to block VEGF signaling and found that this treatment ameliorated the MOG35-55 EAE neurological score by reducing expression of several angiogenic markers Egfl7, Ecm1, Serpine1, and Emcn, as confirmed by both in situ hybridization and computational analysis of scRNA-seq data. Immune profiling of spleens and spinal cords by flow cytometry did not show changes in immune cell activation in bevacizumab-treated mice relative to IgG controls, indicating that the protective effects of VEGF blockade are not due to defects in the initiation of the immune response. Finally, in Chapter 5, I summarize the major findings of my dissertation and propose a model for the mechanisms by which neo-angiogenesis contributes to pathology in MS/EAE. I also present several future avenues of research that can be pursued to further our understanding of the molecular and cellular changes underlying pathogenic angiogenesis and its role in MS/EAE. While most current disease-modifying MS therapies aim to reduce inflammation and infiltration of immune cells into the CNS, these findings may lead to development of additional potential therapeutics that may reduce pathogenic neo-angiogenesis in order to alleviate long-term neurological deficits in MS. Additionally, since postcapillary venules and veins are the major sites of immune cell infiltration, BBB damage and neo-angiogenesis in EAE, the findings of this study suggest that development of treatment modalities that target venous ECs with anti-angiogenic compounds may be more effective in inhibiting the growth of pathogenic neovessels than therapies directed against the entire endothelium.

No description available.

Thesis (PhD)--Stellenbosch University, 2001 / ENGLISH ABSTRACT: The eye is that biological instrument which conveys the light of the external world into the inner world of the mind, wherein we receive the miraculous gift of vision. So precious is this gift, that Science must search for ways to keep this portal clear for the flow of light. Indeed, Science is called upon to “make war upon the bloody tyrant, Time.” (Shakespeare W. Sonnet No. 16). For, in the course of ageing, the lens grows cloudy and cataractous. In this battle between Science and Time, we are fortunate to live in an era in which Science is uncovering the molecular basis for the various obstacles to vision. The question arises, whether or not, the ruinous hand of time can be stayed. Due to unrelenting, progressive lens opacification, most of the elderly are destined to be subjected to loss of vision and with passage of time, even blindness. Globally the cataract surgery rate is inadequate to keep pace with the ever growing demand on financial and human resources created by the cataract problem. An immense challenge therefore is directed to primary eye care: “Can cataract be prevented or can its onset at least be postponed?” This laudable ultimate aim can only be achieved once the etiology of cataractogenesis is well understood. This dissertation seeks to examine two previously unrecognized etiological aspects that, if correctly understood and managed, have the potential to achieve preventive ophthalmological goals that may indeed help to stay the ‘ruinous hand of time’. The first aspect deals with the role of lipids and was examined using a study group of dyslipidemic subjects. The first part of the study concluded that dyslipidemic patients develop cortical lens opacities more frequently and at an earlier age than the normal population, and that cortical lens opacities should be regarded as one of the most reliable clinical signs of dyslipidemia. Furthermore, an extremely strong correlation was found to exist between low HDL Cholesterol levels and the development of opacities. Below a HDL-Cholesterol level of 1,5mmol/l, subjects had more than seven-fold higher risk of falling in the lens opacity subgroup than those with HDLCholesterol levels above 1,5mmol/l. An equally strong correlation was demonstrated between high (>5) LDLHDL ratios and the development of lens opacities. Subjects with a LDL:HDL-C ratio below 5 possessed a 2.35 times greater risk of having lenticular opacities than the group with a LDL:HDL-C ratio greater than 5. The prevention or retardation of dyslipidemia associated lens opacities is therefore possible, provided patients with a genetic predisposition are detected early and their blood lipids managed adequately. The second aspect deals with the relationship between age related cataracts and the acetylation status of the individual. This study compellingly submits that the slow acetylator pheno- and genotype may be regarded as a genetic indicator of risk for age related cataract. The ability accurately to classify a patient genotypically and phenotypically, may henceforth be useful in health counseling since, if an individual is identified as being a slow acetylator, additional preventative and precautionary measures may be taken, i.e. the prevention of UVexposure to the eye and caution with the ingestion of xenobiotics like caffeine, commercial dyes, food preservatives, and drugs. Furthermore, such a finding should be taken into account in the long term therapeutic management of glaucoma, with special regard to carbonic anhydrase inhibitors which are sulphonamide-related drugs and totally dependent on the N-acetyltransferase pathway for metabolism. These drugs may accumulate in the slow acetylator over time and exert toxic effects intra-ocularly, conceivably including cataractogenesis. The search for genetic and metabolic mechanisms that may contribute to human cataractogenesis should be pursued with great enthusiasm. This endeavour may help Science to achieve its primary objective, ablate the effects of Time and really aid in preventing cataracts in man. / AFRIKAANSE OPSOMMING: Die wondergawe van visie word vir ons moontlik gemaak deur die oog wat as biologiese instrument die lig van die buitewereld inlaat na die binnewereld van die brein. So kosbaar is hierdie gawe dat die Wetenskap deurgaans moet poog om die poort oop te hou. Inteendeel, die Wetenskap word gemaan deur Shakespeare in sy Sonnet nommer 16 om “oorlog te maak teen die bloeddorstige tiran, Tyd”. Soos ‘n mens ouer word, word die lens dof en ‘n katarak mag vorm. Ten spyte van hierdie stryd tussen ‘Wetenskap’ en ‘Tyd’ leef ons in die gelukkige era waarin die Wetenskap meer en meer leer van die verskeie obstruksies tot visie. Die vraag ontstaan of die rinnewerende hand van ‘Tyd’ gestuit sal kan word. Vanwee ongenaakbare, progressiewe lensvertroebeling is die meeste bejaardes bestem om aan visie verlies, en met verloop van tyd selfs blindheid, te ly. Die wereldwye katarakchirurgie tempo is nie voldoende om by te hou by die immergroeiende finansiele en mannekrag eise wat deur die katarak probleem gestel word nie. Daar word dus ‘n reuse uitdaging aan primere oogsorg gestel naamlik: “Kan katarakte nie eerder voorkom of die aanvang daarvan ten minste uitgestel word nie?” Hierdie prysenswaardige doelwit kan nie bereik word alvorens die etiologie van kataraktogenese goed verstaan word nie. Hierdie tesis ondersoek twee voorheen onerkende etiologiese aspekte wat, indien hulle korrek verstaan en hanteer word, beslis die potensiaal het om die gemelde voorkomende doelwitte te bereik en sekerlik te kan bydrae om die rinnewerende hand van Tyd te stuit. Die eerste aspek spreek die rol van lipiede aan deur te kyk na 'n studiegroep van dislipidemiese persone. Die eerste deel van die studie kom tot die gevolgtrekking dat dislipidemiese pasiente kortikale lens opasiteite meer dikwels en op ‘n vroeer ouderdom ontwikkel as die normale populasie en dat sulke opasiteite beskou moet word as een van die mees betroubare kliniese tekens van dislipidemie. Daar is ook ‘n baie sterk korrelasie gevind tussen lae HDL cholesterol vlakke en die voorkoms van opasiteite. Persone in die studie met ‘n HDL cholesterol vlak laer as 1,5mmol/l het ‘n sewe keer hoer kans gehad om in die lensopasiteit subgroep te val as die met ‘n HDL cholesterol vlak laer as 1,5mmol/l. ‘n Sterk korrelasie tussen ‘n hoe (>5) LDLHDL verhouding en die voorkoms van lens opasiteite is ook gevind. Persone met ‘n LDLHDL verhouding >5 het ‘n 2.35 maal groter risiko gehad om lensopasiteite te he as die met ‘n LDL:HDL verhouding van <5. Die voorkoming of vertraging van dislipiedemie geassosieerde lens opasiteite is dus moontlik, solank persone met ‘n genetiese geneigdheid daartoe vroeg ontdek en hulle bloedlipiede voldoende beheer word. Die tweede deel van die tesis handel oor die verhouding tussen ouderdoms verwante katarakte en die asetilasie status van die individu. Met oortuiging kom hierdie studie tot die gevolgtrekking dat die stadige asetilator fenoen genotipe as 'n genetiese merker vir ouderdoms verwante katarakte beskou moet word. Die vermoe om ‘n individu beide genotipies en fenotipies akkuraat te klassifiseer mag voorts bruikbaar wees in gesondheidsraadgewing. Indien ‘n individu geTdentifiseer is as ‘n stadige asetileerder, kan addisionele voorsorg maatreels getref word soos bv. die voorkoming van blootstelling van die oog aan UV lig sowel as omsigtigheid met die inname van xenobiotika soos kaffei'ene, kleurstowwe, voedsel preserveermiddels en geneesmiddels. Hierdie bevinding moet ook in berekening gebring word in die langtermyn terapeutiese hantering van gloukoom. Die koolsuuranhidrase inhibitore, dikwels gebruik in die behandeling van gloukoom, is sulfonamied-agtige middels en dus totaal afhanklik van die N-asetieltransferase pad vir hulle metabolisme. Hierdie middels kan ophoop in die stadige asetileerder en gegewe genoeg tyd, bes moontlik toksiese intra-okulere effekte tot gevolg he. Die soeke na genetiese en metaboliese meganismes wat mag bydra tot menslike kataraktogenese behoort nagestreef te word met groot entoesiasme. Hierdie strewe mag dalk net vir die 'Wetenskap' bystaan om sy primere mikpunt te bereik, die effek van ‘Tyd’ te neutraliseer en te help om katarakte werklik te voorkom.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

Parkinson‟s disease (PD) is the most common neurodegenerative movement disorder. It is characterized by the progressive degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc). Although the etiology of PD remains incompletely understood, emerging evidence suggests that iron homeostasis dysregulation may be involved. A pathological hallmark of PD is the formation of Lewy bodies, intra-cytoplasmic inclusions that are major composed of α-synuclein (α-syn). α-synuclein is encoded by the SNCA gene. It is generally believed that α-synuclein aggregation is a main pathogenic feature and the cause of PD. Previous in vitro studies have provided direct evidence showing that iron could interact with α-synuclein and facilitate its aggregation. Nevertheless, the exact role of iron in the pathogenesis of PD is still inconclusive, and so far no studies have proved the interaction between iron and α-synuclein in vivo. / Here, based on a Drosophila model, we tested the hypothesis that the interaction between iron and α-synuclein accumulation accelerates the pathogenesis of PD, and that restoring brain iron homeostasis provides neuroprotective effects against PD. In our present studies, two groups of Drosophila, including w¹¹¹⁸ control and mutant α-synuclein A53T Drosophila were cultured under normal- (normal medium) and high-iron diet (medium added with 30mM ferric ammonium citrate (FAC)) for up to 30 days. During chronic iron treatment, startle-induced negative geotaxis assay was conducted every ten days to test the locomotor ability in the flies. After that, whole-mount immunostaining was used to assess dopaminergic neuronal survival. These flies were also collected and subjected to the quantification of brain iron content for the characterization of the brain iron content status. Furthermore, quantitative real-time PCR and western-blot analysis were conducted to investigate the amount of various α-synuclein conformations. / In the first part, we observed that α-synuclein A53T fly exhibited age-related increase of brain iron content compared with age-matched control. These were accompanied by shorter life-span, locomotor dysfunction, and TH-positive neuronal loss in PPM1/2 and PPM3 cluster. Meanwhile, we have demonstrated that neuronal toxicity and motor deficits were associated with increased proteinase K resistant, insoluble α-synuclein rather than the total amount of protein level. The insoluble α-synuclein was regarded as α-synuclein aggregation. / In the second part, we found that in α-synuclein A53T fly, excessive iron uptake aggravated locomotion defects and led to specific TH positive neuronal loss in cluster PPM3 after 30 days of iron treatment. Moreover, the excessive iron-induced neurological toxicity and motor dysfunction were also associated with increased α-synuclein aggregation. Overall, these two sets of results suggest that abnormal up-regulation of brain iron content may be associated with α-synuclein, and contribute to the pathogenesis of PD through α-synuclein aggregation-dependent mechanisms. / In the third part, we further explored the potential neuroprotective effect of restoring brain iron homeostasis in PD. We made use of genetic modification to manipulate iron-transport protein DMT1 expression, in turn to identify the protective effect of decreasing brain iron content in α-synuclein Drosophila model. Our present results proved that inactivation of Malvolio in α-synuclein A53T fly can suppress the increase of brain iron contents, and can also prolong life span, partially ameliorate locomotion deficits, and attenuate TH positive neuronal loss in α-synuclein A53T fly. In addition, these beneficial effects might occur through the inhibition of α-synuclein aggregation in α-synuclein A53T fly. Consequently, this result implicates that reducing brain iron by inactivation of iron up-take protein DMT1 can inhibit α-synuclein aggregation and provide beneficial effect on DA neuronal survival in PD model. / In conclusion, we demonstrated that : (1) abnormal up-regulation of brain iron content may be associated with α-synuclein and contributes to the pathogenesis of PD through α-synuclein aggregation-dependent mechanisms; (2) iron uptake protein DMT1 may serve as a potential therapeutic target for alleviating aberrant iron accumulation and retards the progression of neurodegeneration in PD. / 帕金森氏病（PD）是最常見的神經退行性疾病之一，其病理學特徵是黑質緻密部（SNpc）的多巴胺能神經元退行性變性。和，其中α-突觸核蛋白（α-synuclein）是Lewy小體的主要成分。雖然帕金森氏病的發病機制仍不十分清楚，隨著研究的進展，越來越多的證據表明鐵穩態失調可能是其中一個重要的致病因素。細胞內Lewy小體聚集物的形成是帕金森病的一個病理標誌物，其主要的成分是α-synuclein蛋白。α-synuclein是由SNCA基因編碼的蛋白。其聚集通常被認為是帕金森病的一個主要的病理特徵，同時也是導致帕金森病的一個原因。之前的研究證據表明，在體外實驗中，鐵能夠與α-synuclein相互作用並促進α-synuclein的聚集。然而，鐵和α-synuclein的相互關係在帕金森病的發病機制中的確切作用仍不十分確定。 / 我們的課題是基於果蠅模型來驗證腦鐵增加導致的α-synuclein聚集加速了帕金森病的病理進程，且恢復腦鐵平衡可以保護帕金森病人多巴胺能神經元的假說。在我們目前的研究中使用了兩組不同基因型的果蠅，其中包括w¹¹¹⁸對照組和突變體α-synA53T果蠅。兩組果蠅分別同時餵養正常食物（正常培養基）和高鐵食物（30mM檸檬酸鐵銨（FAC））。經過30天的慢性鐵處理，分別收集不同組的果蠅並測定其腦鐵含量用於對不同組果蠅的腦鐵含量進行定量分析。在進行鐵處理的30天期間，每隔10天進行一次趨地性行為學實驗用於評價不同組果蠅的運動能力。行為學實驗過後，可使用整腦免疫染色法來觀察果蠅多巴胺能神經元的存活情況。此外，還运用实时定量PCR和蛋白免疫印跡分析法來檢測不同組果蠅體內α-synuclein mRNA和蛋白的表達情況。 / 在第一部分的實驗中我们發現，與年齡相當的對照組相比，α-synA53T果蠅表現為明顯增高的腦鐵含量，並伴隨著壽命短，運動功能障礙以及PPM1/2和PPM3多巴胺能神經元簇的TH-陽性神經元丟失。同時我們發現，α-synA53T果蠅的神經元的毒性和運動障礙與α-synuclein的蛋白總量無關，而可能與在蛋白酶K中穩定的不溶性α-synuclein蛋白的增加相關，這部分α-synuclein的增加被認為與α-synuclein聚集有關。 / 此外在第二部分實驗中我們還發現，α-synA53T果蠅經過30天鐵處理後會加劇其運動功能障礙，並導致更嚴重的PPM3多巴胺能神經元簇的TH-陽性神經元丟失。並且這種由腦鐵含量增加而導致的神經毒性和運動功能障礙也與α-synuclein聚集增加有關。這兩部分的實驗結果表明，腦鐵含量異常增加調節α-synuclein聚集可能與帕金森病的發病機制有關。 / 在第三部分實驗中，我們進一步探討維持腦鐵穩態在PD中潛在的神經元保護作用。我們利用遺傳學手段調節鐵轉運蛋白DMT1的表達來研究腦鐵含量減少對α-synuclein果蠅的保護作用。目前的結果表明，降低α-synA53T果蠅中DMT1同源基因-Malvolio的表達可以抑制隨著年齡增加而顯著增加的腦鐵含量，並且可以延長果蠅的壽命，部分改善α-synA53T果蠅的運動功能障礙，以及完全減輕了TH陽性神經元的丟失。這些保護作用可能是由於抑制了α-synA53T果蠅中α-synuclein的聚集。因此，這些結果顯示，通過降低DMT1的表達來減少腦鐵含量DMT1可以抑制α-synuclein蛋白聚集並對神經元有保護作用。 / 綜上所述，我們的實驗結果證明了：（1）腦鐵含量異常增加調節α-synuclein聚集可能與帕金森病的發病機制有關; （2）鐵攝取蛋白DMT1可作為一個潛在的選擇性鐵螯合劑治療靶標來減緩帕金森病神經退行性進程。 / Zhu, Zhoujing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 155-181). / Abstracts also in Chinese. / Title from PDF title page (viewed on 03, October, 2016). / Zhu, Zhoujing. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.