Neisseria meningitidis is an important cause of septicaemia and meningitis, yet can be found colonising the nasopharynx of up to 20% of healthy individuals. The aim of my work was to provide detailed characterisation of the cellular location of meningococcal carriage and subsequently select suitable models to investigate the molecular and cellular basis of the mechanisms by which the bacterium interacts with the human upper respiratory tract. This is the fundamental microbial-host interaction underlying the commensalism of Neisseria meningitidis. A survey of meningococcal carriage in tonsillar tissue was undertaken using IHe techniques that detect PorA, a protein unique to Neisseria meningitidis. This showed that carriage rates are higher than those described with nasopharyngeal swabbing, and that the bacterium occupies a site deep to the epithelium in the carrier state. To identify genes that are required to reach sub-epithelial sites, air-interface organ culture models were used to screen bacteria subjected to signature tagged mutagenesis. Ten potentially colonisation deficient mutants were isolated and further analysed. This is the first time that such a screen has been undertaken in tissue of human origin. Additionally, homologues of two genes essential for intracellular survival in Legionella pneumophila (macrophage infectivity potentiator and an unknown virulence protein) were identified in Neisseria meningitidis and mutants containing specific genetic deletions constructed.

No description available.

The purpose of this study was to examine the phenomenon of aging at the cellular level in vitro. Some of the biological mechanisms underlying the aging process, which may invoke a change at the level of the DNA were studied. Morphological changes were analysed with phase-contrast microscopy and functional changes by the use of tritiated thymidine in combination with autoradiography as well as by cell treatment with colchicine. A method is described for obtaining "aged" or "old" cells in vitro. In the human cells (human embryonic kidney) as well as the rat, mouse, and Syrian hamster cells, morphological changes in vitro are basically the same as aging progresses. These include transformation to a polygonal, epithelial -1ike shape; binucleation; an accumulation of "age pigments" around the nucleus; the appearance of ragged edges of the cell membrane; an increase in the overall cell size; and a loss of a regular (often parallel) orientation to adjacent cells. The mitotic rate and DNA-synthesizing capacity in "young" and "aged" cells were examined using autoradiography and cell treatment with colchicine. Evidence is presented that DNA-synthesizing aged cells are non-proliferating while DNA-synthesizing young cells are mitotically active. The significance of DNA-synthesis in non-dividing "aged" cells is discussed. The number of population doublings (generation times or cell divisions) that it takes hamster and mouse cells to age in vitro was also investigated. Thirteen and six cell generation times were found to cause hamster and mouse cells to age with a loss of proliferative capacity. The effect of various molarities of 4-nitroquinoline-1 oxide (4-NQO), on DNA of aged cells, which results in an unscheduled DNA-repair synthesis, was studied using autoradiography. It appears that an aged cell responds to these concentrations in much the same way as young cells; however, there does seem to be a slightly greater sensitivity to toxic doses of 4-NQO in aged cells. Autoradiographic studies also revealed that the duration of DNA-repair is the same in both aged and young cells, but the former appear to have a decreased capacity to repair damage to the DNA of the pretreated cells caused by the 4-NQO. The significance of this apparent decrease in the DNA-repair capacity of "aged" cells is discussed. Mouse cells, aged in vitro, were exposed to human adenovirus type 12 (strain Huie). Evidence is presented that this external agent stimulated these aged cells to increase DNA-synthesis and also pushed them into mitosis (for at least one cell division). The possibility that this might be an accountable mechanism for the observation of an accumulation of mutations in aged cells is evaluated. Aged cells were examined for frequencies and types of chromosome aberrations after exposure to adenovirus type 12. Among the most common are chromatid breaks and double fragments. As well, old cells exhibited a much higher frequency of chromosome aberrations than young cells after viral exposure. A comparison of this in vitro system of cell aging with an in vivo system is presented. The application of all of the aforementioned results and observations concerning the cellular aging process to the problem of carcinogenesis and neoplasia is emphasized. / Science, Faculty of / Zoology, Department of / Graduate

The cells of the licorice plant, Glycyrrhiza glabra, were cultured as a "single cell" suspension. Their growth behaviour, yield and metabolic products were studied. The suspension cultures of the licorice plant were established from the friable calluses obtained from the radicle, cotyledon and hypocotyl of the germinated seeds. The single cells, regardless of their origin showed little difference in cell size and morphology. After an apparent adjustment to the medium, the cells required 11-13 days of incubation to reach the maximum cell yield of 1.2 gm/100 ml medium, dry weight. During the growth period, the pH of the growth medium decreased from pH 5.6 to pH 4.7 in the first few days and then increased to about pH 6. A level of 10% coconut milk in PRL-4-CM medium was found to support good cell growth; the lower the coconut milk level, the longer the growth period required to reach the maximum cell yield. It was also found that 0.57% yeast extract could be used to replace the coconut milk in the PRL-4-CCM medium. The metabolites detected and examined in the licorice single cell suspension culture included a volatile apple aroma, a polysaccharide pectin-like material, steroids and triterpenoids. The analyses of the licorice cell volatile apple aroma found under anaerobic conditions indicated the presence of ethanol and some related esters. The monosaccharides found in the pectin-like polysaccharide hydrolysate were glucose, fructose, galactose, arabinose, xylose, galacturonic acid and glucuronic acid. The pectin-like material in the cell preparations reached a maximum yield of 1.1 mg/ml after one month of growth. Glycyrrhizinic acid, the common licorice constituent found in the root, could not be detected in the suspension cultures. However, several other related compounds which gave typical steroid and triterpenoid reactions were found. Sorbitol and fructose were found to be the two major sugars which accumulated in free form in the licorice cell medium. / Land and Food Systems, Faculty of / Graduate

Die Erzeugung von klinisch in der plastischen und rekonstruktiven Chirurgie nutzbarem Fettgewebe stellt einen sehr wichtigen Aspekt in aktuellen Arbeiten des Tissue Engineerings, also der Erzeugung von spezifischem Gewebe aus Spenderzellen dar. Sollte es gelingen, aus patienteneigenen Zellen wieder neues Gewebe zu züchten, so würden daraus eine Fülle neuer Behandlungsmöglichkeiten für Gewebedefekte resultieren. In einer Vorgängerarbeit zu der vorliegenden Arbeit konnte gezeigt werden, dass die Adipogenese in vivo von Fettgewebe aus Vorläuferzellen, den Präadipozyten, durch geeignete Methoden der Vorkultivierung in vitro beeinflusst werden kann. Die Unterschiede in der Vorbehandlung lagen in einer Induktion der Differenzierung der Präadipozyten bei gleichzeitigem Stopp der Proliferation und einer anschließenden verschieden langen Ausdifferenzierungsphase der Zellen in vitro im Brutschrank. Die resultierenden Konstrukte wurden in jeweils drei Mäuse in vier Gruppen implantiert und nach 1, 5, 12 und 24 Wochen entnommen und untersucht. Während die Präadipozyten von Gruppe 1 keine Induktion erfuhren, erfolgte diese bei den anderen drei Gruppen. Die Konstrukte der Gruppe 2 wurden dann bereits nach 2 Tagen der Induktion der Präadipozyten implantiert, die Konstrukte der Gruppe 3 blieben zur Differenzierung noch 7 Tage, die der Gruppe 4 noch 33 Tage im Brutschrank, bevor sie in die Versuchstiere eingebracht wurden. Ziel der vorliegenden Arbeit war es zunächst, an den Gewebekonstrukten der Vorgängerarbeit eine histomorphometrische Analyse der resultierenden Adipozyten in vivo über die Zeit durchzuführen, um eine detaillierte Beurteilung des Verlaufs der Fettgewebeentwicklung anhand resultierender Zellzahlen darzustellen. Hierfür wurden die Gewebedünnschnitte der Mäuse nach einer HE-Anfärbung mikroskopisch untersucht und die Zellzahlen resultierend jeweils aus unreifen und reifen Adipozyten histomorphometrisch quantifiziert. Die Unterscheidung erfolgte mittels einer Größenzuordnung, wobei Zellen kleiner 20 µm Durchmesser den unreifen und Zellen größer 20 µm Durchmesser den reifen Adipozyten zugeordnet wurden. Aus der quantitativen Analyse mittels Histomorphometrie ergab sich, dass in allen Konstrukten die Zahlen an Zellen der den unreifen Adipozyten zugeordneten Größenordnung von kleiner als 20µm tendenziell während der gesamten Zeit in vivo klein bleibt. Die Zellzahlen resultierend aus großen Zellen mit einem Durchmesser mehr als 20µm, die den reifen Adipozyten zugeordnet wurden, steigen dagegen in allen Proben leicht an, wobei die Konstrukte der Gruppe 4 den absolut höchsten Wert aufwiesen. In der HE-Anfärbung ist demgemäß in Gruppe 4 eine Vielzahl reifer Adipozyten zu erkennen. Das zweite Ziel dieser Arbeit war es, durch Anfärbung charakteristischer Proteine der extrazellulären Matrix mittels markierter Antikörper und einer anschließenden immunohistochemischen Analyse des Verlaufs der Signalintensität dieser markierten Komponenten in der EZM die Adipogenese mittels Analyse der entstehenden Gerüstproteine zu verfolgen. Hierfür wurde durch eine umfangreiche immunohistochemische Analyse die Bildung der Kollagene I, IV und VI sowie von Laminin als Bestandteile der EZM analysiert und damit die Art und der Umfang der entstandenen extrazellulären Matrix während der Adipogenese qualitativ beurteilt. Die Fluoreszenz-Bilder der Proben nach den jeweiligen Gruppen und Wochen in vivo zeigen einen deutlichen Hinweis im Sinne der Bildung von Fettgewebe in den Gewebe-Konstrukten der Gruppe 4. Während in den Gruppen 1 und 2 fast durchweg faserartige Bindegewebsstrukturen, verbunden mit den entsprechenden eher fibrillärem Aussehen der Signale für die untersuchten Kollagene I, IV, VI und für Laminin gefunden werden konnten, zeigen die Konstrukte der Gruppe 3 und insbesondere von Gruppe 4 in den Fluoreszenz-Abbildungen deutlich ausgeprägtere, netzartig ausgebildete Strukturen. Aus den Resultaten der vorliegenden Arbeit kann demnach geschlossen werden, dass die Art der Vorkultivierung eine spätere Adipogenese eindeutig beeinflussen kann. Eine längere Inkubationszeit nach erfolgter Induktion der Präadipozyten zur Förderung der Reifung zu Adipozyten vor der Implantation fördert die Bildung einer höheren Anzahl von Adipozyten und die Ausbildung einer charakteristischen EZM. Diese Erkenntnisse eröffnen für zukünftige Arbeiten die Möglichkeit, durch die weitere Optimierung der Vorkultivierung, verbunden mit einer eventuell noch besseren Überlebensrate der ursprünglich eingebrachten Zellen, die Herstellung von klinisch geeigneten Konstrukten aus Fettgewebe weiter voranzutreiben. / The generation of adipose tissue that can be clinically used in plastic and reconstructive surgery is a very important aspect of current tissue engineering work, i.e. the generation of specific tissue from donor cells. Should it be possible to grow new tissue from the patient's own cells, it would result a plethora of new treatment options for tissue defects. In a previous work to the present work it was shown that the adipogenesis in vivo of adipose tissue from precursor cells, the preadipocytes, can be influenced by suitable methods of preculturing in vitro. The differences in the pretreatment were an induction of the differentiation of the preadipocytes with simultaneous stop of the proliferation and a subsequent differentiation phase of the cells in vitro in the incubator. The resulting constructs were implanted in four groups of three mice each and removed and examined after 1, 5, 12 and 24 weeks. While the preadipocytes of group 1 did not experience induction, this did occur in the other three groups. The constructs of group 2 were then already implanted after 2 days of the induction of the preadipocytes, the constructs of group 3 remained in the incubator for 7 days for differentiation, those of group 4 for 33 days before they were introduced into the test animals. The aim of the present work was initially to carry out a histomorphometric analysis of the resulting adipocytes in vivo over time on the tissue constructs of the previous work in order to present a detailed assessment of the course of adipose tissue development based on the resulting cell counts. For this purpose, the thin tissue sections of the mice were examined microscopically after HE staining and the cell counts resulting from immature and mature adipocytes were quantified histomorphometrically. The differentiation was made by means of a size assignment, with cells smaller than 20 µm in diameter being assigned to immature and cells larger than 20 µm in diameter being assigned to mature adipocytes. The quantitative analysis by means of histomorphometry showed that in all constructs the number of cells of the order of magnitude of less than 20 µm assigned to immature adipocytes tends to remain small during the entire time in vivo. In contrast, the cell counts resulting from large cells with a diameter of more than 20 µm, which were assigned to the mature adipocytes, increased slightly in all samples, the constructs of group 4 exhibiting the absolute highest value. Accordingly, a large number of mature adipocytes can be seen in group 4 of the HE staining. The second aim of this work was to follow the adipogenesis by staining characteristic proteins of the extracellular matrix by means of labeled antibodies and a subsequent immunohistochemical analysis of the course of the signal intensity of these labeled components in the ECM by analyzing the resulting scaffold proteins. For this purpose, the formation of collagens I, IV and VI as well as laminin as components of the ECM was analyzed through an extensive immunohistochemical analysis and thus the type and extent of the extracellular matrix formed during adipogenesis was qualitatively assessed. The fluorescence images of the samples after the respective groups and weeks in vivo show a clear indication of the formation of adipose tissue in the tissue constructs of group 4. While in groups 1 and 2 almost all fibrous connective tissue structures, connected with the corresponding rather fibrillar appearance of the signals for the examined collagens I, IV, VI and for laminin could be found, the constructs of group 3 and in particular of group 4 show clearly more pronounced, network-like structures in the fluorescence images. From the results of the present work it can be concluded that the type of pre-cultivation can clearly influence later adipogenesis. A longer incubation period after induction of the preadipocytes to promote maturation to adipocytes before implantation promotes the formation of a higher number of adipocytes and the development of a characteristic ECM. These findings open up the possibility for future work to further advance the production of clinically suitable constructs from adipose tissue through the further optimization of the preculture, combined with a possibly even better survival rate of the originally introduced cells.

INTRODUCTION: Cephalometric soft tissue findings have shown correlation with pharyngeal width. Facial photographic analysis of patients with Obstructive Sleep Apnea (OSA) shows an increase in width and flatness of the midface. However, three-dimensional facial soft tissue morphology of children with OSA has not been studied. OBJECTIVE: The objective of the current study was to evaluate the association between facial morphology, upper airway volume, Polysomnography (PSG), and Pediatric Sleep Questionnaire (PSQ) findings in children with OSA versus controls. MATERIAL AND METHODS: The sample included de-identified pre-treatment Cone-beam Computed Tomography images, PSG and PSQ results of 36 children (mean age 6.8 ± 2.8) from one pediatric dental practice. Three-dimensional facial soft tissue landmarks were digitized using Mimics v.20 software. Upper airway volume was segmented into right nasal cavity (RNC), left nasal cavity (LNC), nasopharynx (NP), oropharynx (OP), and hypopharynx (HP). Apnea Hypopnea Index (AHI), Respiratory Disturbance Index (RDI) scores and Pediatric sleep questionnaire (PSQ) values were correlated with soft tissue measurements (a modified Farkas anthropometric analysis) and upper airway volumes using Pearson’s correlation. Student’s T-test was used to evaluate the difference between facial soft tissue measurements of children with obstructive sleep apnea (OSA) versus the control group. RESULTS: Experimental versus control: Polysomnography findings: Apnea/Hipopnea Index and Respiratory Disturbance Index were statistically higher in obstructive sleep apnea children compared to controls (p=<.0001, 0.0001), and lowest oxygen percentage SpO2 was significantly lower (p=0.006). Airway volume findings: Right nasal cavity was statistically larger in obstructive sleep apnea children compared to controls (p=0.04). Soft tissue findings: Exocanthus right to midsagittal plane, Exocanthus right and Exocanthus left, and Soft tissue orbitale right to midsagittal plane were smaller in obstructive sleep apnea children compared to controls (p=0.01, 0.02, 0.03). Experimental group results: Transverse. Nose: Bialar distance was positively correlated to right nasal cavity and nasopharynx, and negatively correlated to hypopharynx, Apnea/Hipopnea Index, and Respiratory Disturbance Index. Lips: Chelion Right and Left, and Crista Philtri Right and Left were positively correlated to NasoPharynx. Anteroposterior. Most of nose and lips measurements were positively correlated to Right Nasal Cavity and negatively correlated to Respiratory Disturbance Index and low oxygen percentage SpO2. Vertical. Nose measurements were positively correlated to NasoPharynx. Lips measurements were positively correlated to NasoPharynx and OroPharynx and negatively correlated to low oxygen percentage SpO2. Control group result: Transverse. Nose: Nostril Base Right to midsagittal was positively correlated to NasoPharynx and average oxygen percentage SpO2 and negatively correlated to Respiratory Disturbance Index. ProNasale to Nostril Base Right and Nostril Base Left was negatively correlate to Pediatric Sleep Questionnaire. Lips: Crista Philtri Right to midsagittal and Chelion Right to midsagittal plane were positively correlated to NasoPharynx and average oxygen percentage SpO2. Anteroposterior. Nose measurements were positively correlated to Right Nasal Cavity, NasoPharynx, OroPharynx, and HypoPharynx and negatively correlated to Pediatric Sleep Questionnaire. Lips measurements were positively correlated to Right Nasal Cavity, NasoPharynx. And OroPharynx. Vertical. Most nose and lips measurements were positively correlated to Right Nasal Cavity, NasoPharynx, OroPharynx, and HypoPharynx, and negatively correlated to Apnea/Hypopnea Index and Respiratory Disturbance Index. CONCLUSION: It can be concluded that for the experimental group wider faces at the level of the eyes, nose and lips indicated increased upper airway volumes, decreased Polysomnography, and Pediatric Sleep Questionnaire values. Moreover, more forward position of the nose and lips in relation to the coronal plane were linked to increased nasal airway volume and decreased Polysomnography values. Finally, long-faced individuals displayed higher volume of the upper airway and decreased oxygen saturation levels. In regards to the control group, anteroposterior measurements positively correlated to all airway compartments and negatively correlated to Pediatric Sleep Questionnaire values. Vertically, longer faces exhibit larger airway compartments and decreased Polysomnography values.

The amniotic membrane is a lining along the inner aspect of the placenta that envelops a developing embryo (then fetus). This component is critical for the adequate growth and nutrition of the fetus and can greatly impact the viability of the fetus. This role in development has led scientists to explore its post-delivery uses in regenerative medicine. Specifically in this paper, current literature was reviewed to determine the applicability of amniotic membranes to neural tissue engineering. The amniotic membrane has been greatly characterized with respect to immune response (including inflammatory effects) and microbial influence. These preliminary characterizations of the amniotic membrane and its components (i.e. stem cells) demonstrated a promising future for clinical implementation. Some fields, such as cardiovascular and orthopedic research, have begun projects using either the amnion-derived stem cells or amniotic membrane as a central element in their research. Both elements have received extensive praise for their versatility and relatively easy implementation into multiple organs and systems in the body. In each of these systems, the amniotic membrane retained its optimal antimicrobial and anti-immunogenic characteristics. After researching the current applications, it was apparent that amniotic membranes could have a place in the future of neural tissue engineering whether it be axillary components (cerebral vessels or surrounding bone) or direct regeneration of nerves. The largest impediment was the lack of basic science understanding in neuroscience. As the specific mechanisms of normal brain behavior and disease states are uncovered amniotic membranes can be added to the pre-clinical testing for the neurological sciences. Similar to the other fields of medicine, amniotic membranes will be specifically useful due to their ability to not evoke an immune response, thereby mitigating the possibility of rejection and infection in the central nervous system. These elements are critical to the research in neurology. Overall, amniotic membrane research would be very valuable to neuroscientists and physicians when exploring the future of neural tissue engineering

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Castor (Ricinus communis L.) is a high-yielding oilseed crop native to tropical Africa. The seed contains ~60% oil by weight, yielding approximately 1,200 kg of oil per hectare. The oil is composed of ~90% ricinoleic acid, a unique hydroxylatty acid. Its unique composition provides castor oil with distinctive characteristics important for industrial use. Unfortunately, this valuable oilseed has not been widely cultivated in the United States since 1972, due in part to the presence of ricin in the seed. Ricin is a highly toxic lectin found in the endosperm of mature castor seed. This project sought to silence ricin production through the introduction of an RNAi element into the castor genome. The RNAi vector (pC1-RKO) containing a segment of ricin mRNA and its inverted repeat separated by a chalcone synthase A intron from pFGC5941 enclosed in a pCambia1301 backbone was created, verified via sequencing, and transformed into Agrobacterium tumefaciens for castor transformation. Fungal contamination was a serious concern; successful disinfestation used a 10-minute wash with 0.1% mercuric chloride (w/v). Media supplemented with 6-benzylaminopurine generated healthier shoots from embryo axes dissected from mature seed compared to thidiazuron-treated mesocotyls dissected from mature seed. Short treatments of thidiazuron on 6-benzylaminopurine initiated shoot cultures showed greater shoot proliferation on embryo axes dissected from mature seed. Rooting occurred with incubation on half-strength medium containing naphthaleneacetic acid or indole-3-butyric acid; however, naphthaleneacetic acid produced hardier roots which better survived acclimatization. Inoculation of embryo axis explants after 2 days pre-culture improved survivability. Likewise, transformations using A. tumefaciens cultures of 0.5 O.D.600 and lower did not lead to downstream bacterial contamination. The pCambia1304 vector was used as a test plasmid for refinement of the transformation protocol. Of the 870 pCambia1304 inoculation explants, 2 survived hygromycin screening and showed gusA activity. Of the 2,500 pC1-RKO inoculated explants, 6 survived hygromycin selection and rooted. Further analysis via PCR, end-point RT-PCR, and Western and dot-blotting showed these to be non-transformed and ricin content unaffected.

Children with congenital heart defects and patients with faulty or failing valves have the need for a suitable aortic heart valve replacement. Current treatment options have several downfalls and heavy investigation is being done into the design of an engineered valve to find an alternative that would alleviate many of these issues. Understanding the physiology of how cells interact in vivo is crucial to the construction of such valve. This study investigates the effect of cyclic strain in aortic valve endothelial cells on the adhesion molecules, PECAM-1, ƒÒ1-Integrin, VE-Cadherin and Vinculin. Experiments found that cyclic strain plays a role in the development of cell/cell and cell/extracellular matrix adhesions and junctions and is extremely important in the pre-conditioning of a tissue engineered construct. Without this strain the new valve would be more susceptible to inflammation, injury or possible failure after being implanted into the patient.