Dissertations / Theses on the topic 'Histology, Pathological'

Eighty normal cheek teeth and 26 normal incisors extracted from 14 donkeys (median age 19 years) at post mortem were anatomically examined including grossly and by computerised axial tomography (CAT) imaging. Decalcified histology was performed on 54 sections from 18 teeth (8 donkeys), undeclacified histology on 16 sections from 7 donkeys and scanning electron microscopy on 10 sections from 10 teeth (3 donkeys). The dental formulae and tooth number was found to be the same as in horses with a higher prevalence (17 %) of canine teeth in female donkeys. A decrease in tooth length, pulp horn length and pulp horn width with age was illustrated, as was an increase in occlusal secondary dentine depth with age, although not all these age changes were statistically significant. Normal histological and ultrastructural features of donkey teeth were identified and found to be similar to equine findings. Enamel was found to be thicker buccally in both maxillary and mandibular cheek teeth. Quantitative measurements of transverse dentine thickness around pulp cavities, dentinal tubule diameters and densities, and enamel prism diameters were made. Left lower incisors (301) were extracted from 7 donkeys and 6 horses for micro-hardness determination of enamel, primary and secondary dentine using a Knoop Hardness indenter. No significant difference between donkey and horse incisor microhardness was demonstrated. Examination of 19 donkey skulls at post mortem examination showed donkeys to have a higher degree of anisognathia (27%) compared to horses (23%). Post mortem dental examination of 349 donkeys (median age 31) demonstrated a high prevalence of dental disease (93%) and in particular cheek teeth diastemata (85%). Furthermore, age was associated with increasing prevalence of dental disease and diastemata. Diastemata were also associated with the presence of other dental disorders and with colic-related death in affected donkeys. Quantitative measurements of 45 diastemata from 16 donkeys showed no difference in the medial and lateral width of diastemata but periodontal pockets were deeper laterally. The definition of valve and open diastemata were confirmed. Pulp exposure, dental caries and periodontal disease were examined in detail (54 skulls) at post mortem. A total of 19 teeth were extracted for further detailed examination as performed in normal anatomy. Clinical dental examinations were performed on 357 donkeys in the U.K. that were selected for age distribution, and the prevalence of dental disease in different age groups was found to increase from 28% in the youngest group (age 0-10 years) to 98% in the oldest group (age > 35 years). An increased prevalence of most dental disorders with age was demonstrated as was an association between dental disease and weight loss, poor body condition score, supplemental feeding and previous episodes of colic. Clinical dental examination of 203 working donkeys in Mexico showed similar types of dental disorders as found in the U.K. study, with dental disease present in 62%, of which 18% required urgent dental treatment. There was a significant association between age groups and dental disease, and age groups and body condition score, but there was no association between dental disease and body condition score. However, body condition score was not associated with supplemental feeding or faecal egg counts either.

Geology
M.S.
In modern organisms the structure and arrangement of bone apatite crystals is dependent on the arrangement of the organic collagen fibers. This is reflected in the formation of different types of bone tissue, such as woven (immature) or lamellar (mature), in pathological versus normal bone, or fast-growing (woven) versus slow-growing (lamellar) tissue. Because the basic physiological processes of fracture healing are similar in extant vertebrates, similar patterns may exist in fossil taxa. The three questions of interest for this study were the following: 1) Do differences exist in modern bone apatite crystallinity between normal and pathologic bone? 2) Are differences between normal and pathologic tissue consistent in both modern and fossil bone? 3) Does the type of bone tissue affect fossilization? In this study, we use histological and x-ray diffraction (XRD) analyses to examine fracture pathologies in pedal phalanges from the theropod dinosaur Allosaurus fragilis, and two modern bird species, Branta canadensis (Canada goose) and Cathartes aura (turkey vulture). Raman spectroscopy analysis was performed on modern birds, but not fossil material. Stable isotope and rare earth elements (REE) analyses were performed on fossil material to determine if there are differences in how pathologic bone fossilizes compared to normal bone. Results from Raman spectroscopy and XRD confirm that pathologic bone is more crystalline than normal bone in both fossil and modern taxa. Stable isotope and REE analyses do not show any difference in fossilization between pathologic and normal bone, suggesting that these techniques are more suitable for examining taphonomic rather than physiological differences.
Temple University--Theses

Most environments impose periodic or stochastic stress on natural populations, which increase susceptibility to diseases. Infection by Myxobolus cerebralis (exotic parasite causing salmonid whirling disease) is strongly influenced by a stream\'s physicochemical attributes and stressors, which may also affect host pathology. Susceptibility to M. cerebralis varies greatly among different species and subspecies of the salmonid host, but little is known about lesion severity or location of infection among the native Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri). In 2002 and 2003 we performed a series of 10-day sentinel cutthroat fry exposures and habitat assessments in various sites of three M. cerebralis-positive tributaries to Yellowstone Lake: the Yellowstone River, Pelican Creek, and Clear Creek. At 90 and 150 days post-exposure, fry were examined by polymerase chain reaction and histology to determine prevalence, severity, and location of infection. The goal was to identify spatiotemporal patterns of infection, and physicochemical features of the streams influencing it, and potentially facilitating parasite invasion and establishment. Results on fish (young and adult) host infection data, environmental attributes, and tubificid host presence/absence data in the study streams were used to develop an ecological risk assessment for parasite establishment and whirling disease in this ecosystem. Results from our qualitative risk ranking systems suggest that the cutthroat trout of the Yellowstone Lake basin are highly susceptible to M. cerebralis infection, with the most severe lesions in cartilage of the cranium and jaws, especially in systems with high water temperatures and ionic content. Our results also suggest that such environmental features are most conducive to parasite establishment, especially in tributaries of the lake basin used by cutthroat trout as spawning and rearing habitats. Thus, this study has implications for both ecology and parasitology as it reveals that environmental components can affect when and where a pathogen resides within the host, and thereby affect manifestation of disease. Recognition of the specific environmental attributes most conducive to parasite establishment, and disease, can increase future diagnostics, detection, and management efforts, strengthening the likelihood of correctly predicting M. cerebralis\' and similar pathogenic invasions and establishment in unsampled sites.

El Linfoma de células del Manto (LCM) es un linfoma B maduro, con una característica sobreexpresión de la proteína nuclear Ciclina D1. Se caracteriza por presentar un curso clínico agresivo, aunque se ha observado que algunos pacientes tienen un curso clínico más indolente. Recientemente se ha observado que el factor de transcripción SOX11 tiene una sobreexpresión específica en este tipo de linfoma. El diagnóstico diferencial entre el LCM y otros tipos de linfoma B puede ser difícil. Un problema es el diagnostico diferencial entre el Linfoma Difuso de Célula B Grande (LDCBG) Ciclina D1 positivo y las variedades pleomórfica y blastoide del LCM. Dado que los pacientes se tratan de forma diferente es importante identificar parámetros que permitan establecer el diagnostico diferencial. Estudios recientes han identificado algunos LCM que de forma peculiar presentan una diferenciación plasmocelular. Recientes trabajos han implicado SOX11 en el bloqueo de la diferenciación B terminal a través de la expresión forzada de PAX5. Así pues estas observaciones permiten postular que la diferenciación plasmocelular y más ampliamente el inicio de la diferenciación B-terminal en LCM se debe producir preferentemente en LCM SOX11 negativos. En esta Tesis doctoral hemos estudiado el papel del factor de transcripción SOX11 en el LCM. La intención era poder encontrar herramientas prácticas que nos ayuden a poder identificar y caracterizar mejor al LCM. En este sentido hemos estudiado el valor de SOX11 y otros parámetros en el diagnostico diferencial entre el LCM y el LDCBG Ciclina D1 positivo y por otra parte hemos analizado la diferenciación B-terminal en LCM y su relación con la expresión de SOX11. En el primer manuscrito de esta Tesis hemos teñido 206 LDCBG para Ciclina D1 e identificamos tres casos (1.5%) positivos. Los tres casos eran SOX11 negativos y por FISH se confirmó la ausencia de aberraciones de CCND1. Se estudiaron 22 LCM, todos expresando Ciclina D1 con 89% de los casos expresando SOX11, una frecuencia estadísticamente significativa en relación al LDCBG Ciclina D1 positivo. Una cohorte separada de 98 LDCBG resulto negativa para SOX11, con solo un caso Ciclina D1 positivo. Este LDCBG Ciclina D1 positivo tampoco presento aberraciones de CCND1 por estudios de FISH. En el segundo manuscrito de esta Tesis hemos investigado el fenotipo de diferenciación terminal de células B en 60 LCM 41 SOX11-positivos y 19 SOX11-negativos. Se observaron células plasmáticas monotípicas y células linfoides con diferenciación plasmocítica expresando Ciclina D1 en 7 (37%) casos SOX11-negativos, pero en ninguno de los 41 casos SOX11-positivos (p<0.001). Los tumores SOX11-negativos tenían más frecuentemente una expresión intensa citoplasmática con restricción de una cadena ligera de inmunoglobulina que los positivos (58% vs 13%) (p=0.001). Similarmente los casos SOX11-negativos tenían una expresión de BLIMP1 y XBP1 significativamente más frecuente que en los casos positivos (83% vs 34% y 75% vs 11%, respectivamente) (p=0.001). Sin embargo, no se observaron diferencias en la expresión de IRF4/MUM1 entre estos subtipos de LCM. En resumen, las conclusiones principales de esta Tesis son que el LDCBG Ciclina D1 positivo es poco frecuente, es negativo para SOX11 o aberraciones de CCND1. Por tanto SOX11 es útil en el diagnostico diferencial entre LDCBG Ciclina D1 positivo y LCM. Además el LCM SOX11-negativo puede ser un subtipo específico de este tumor que se caracteriza por presentar con mayor frecuencia características morfológicas e inmunofenotípicas de diferenciación terminal de células B, que pueden ser facilitadas por la ausencia del factor de transcripción SOX11.
Mantle cell lymphoma (MCL) is characterized by a proliferation of malignant B lymphocytes, presenting an aggressive clinical course, although some patients have a more indolent presentation. The genetic hallmark of this lymphoma is the t(11;14)(q13;q32) leading to the overexpression of Cyclin D1. SOX11 is new and specific diagnostic marker for MCL. Cyclin D1 overexpression can also be seen in other hematological malignancies such as Diffuse Large B Cell Lymphoma (DLBCL). These cases may pose diagnostic challenges and are difficult to differentiate from MCL. In the first study of this Thesis we stained 206 DLBCLs for Cyclin D1 and identified 1.5% positive cases. These DLBCLs showed SOX11 negativity and absence of CCND1 aberrations. Also 22 MCLs were studied, with 89% cases expressing SOX11. A separate cohort of 98 DLBCLs was negative for SOX11, with only one case expressing Cyclin D1. Experimental studies have shown that silencing of SOX11 in MCL cells promotes the shift from a mature B cell into an early plasmacytic differentiation phenotype suggesting that SOX11 may contribute to tumor development by blocking the B-cell differentiation program. In the second study of this Thesis we have investigated the terminal B-cell differentiation phenotype in 60 mantle cell lymphomas. Monotypic plasma cells and lymphoid cells with plasmacytic differentiation expressing Cyclin D1 were observed in 37% SOX11-negative cases but in none of the SOX11-positive MCLs (p<0.001). BLIMP1 and XBP1 expression was also significantly more frequent in SOX11-negative than positive cases (83% vs 34% and 75% vs 11%, respectively) (p=0.001). These results indicate that SOX11-negative MCL may be a particular subtype characterized by more frequent morphological and immunophenotypic terminal B-cell differentiation features that may be facilitated by the absence of this transcription factor. In summary, the main conclusions of this Thesis are that Cyclin D1-positive DLBCLs are rare and negative for SOX11 or CCND1 aberrations. There fourth SOX11 is useful in differentiating Cyclin D1-positive DLBCL from MCL. We also demonstrated that SOX11-negative MCL is a particular subtype of this tumor characterized by more frequent morphological and immunophenotypic terminal B-cell differentiation features.

Thesis (D.Sc.(Odontology))--University of Pretoria, 2003.
"Published work submitted to the University of Pretoria for the degree of Doctor of Science in Odontology (Oral pathology)". Includes bibliographical references. Also available online.

У роботі проведено дослідження структури та функцій патологоанатомічної служби в Україні, її актуальні проблеми на сучасному етапі та органи при проведенні реформ. Проведено аналіз умов надання діагностичних послуг у Науковому центрі патоморфологічних досліджень СумДУ, як однієї зі складових патологоанатомічної служби, та проаналізовано основні показники його діяльності. На основі цього було виявлено основні проблеми та потенційні кризи у організації та управлінні роботи Наукового центру патоморфологічних досліджень. На основі виявлених проблем розроблені програми розвитку для подолання наявних та можливих перешкод в умовах реформування медицини.
В работе проведено исследование структуры и функций патологоанатомической службы в Украине, ее фактические проблемы на настоящем этапе и органы во время реформ. Был проведен анализ условий предоставления диагностических услуг в научном центре патоморфологических исследований СумГУ, как один из компонентов патлогоанатомической службы, и проанализированы основные показатели его деятельности. Исходя из этого, были выявлены основные проблемы и потенциальные кризисы в организации и управлении работой Научного центра патоморфологических исследований. На основании выявленных проблем были разработаны программы развития для преодоления доступных и возможных препятствий в условиях реформы медицины.
A study of the structure and functions of the pathoanatomical service in Ukraine as well as its actual problems at the present stage and organs during reforms have been conducted. The analysis of the conditions for the provision of diagnostic services at the Scientific Center of Pathomorphological Studies of SSU, as one of the components of the pathoanatomical service has been conducted, and the main indicators of its activity have been analyzed. On this basis, the main problems and potential crises in the organization and management of the scientific center of pathomorphological research have been identified. On the basis of identified problems, development programs have been developed to overcome available and possible obstacles in the conditions of medicine reform.

Disease has been identified as a major problem in the aquaculture industry for the welfare of the fish stocked as well as for its economic impact. The number of diseases affecting cultured fish has increased significantly during recent years with the emergence of several conditions that have added to the overall impact of disease on the industry. Frequently, a lack of scientific knowledge about these diseases is compounded by an absence of effective treatment and control strategies. This has been the case with rainbow trout gastroenteritis (RTGE), an emerging disease of rainbow trout (Oncorhynchus mykiss Walbaum). This study investigated several aspects related to its aetiology and control. A retrospective survey of UK rainbow trout farmers was undertaken to ascertain the extent and severity of RTGE in the UK as well as to identify RTGE risk factors at the site level. Participants in this study accounted for over 85% of UK rainbow trout production in 2004. It was found that the total number of RTGE-affected sites had risen from 2 in the year 2000 to 7 in 2005. The disease was only reported from sites producing more than 200 tonnes of trout/year for the table market. Analysis of risk factors associated with RTGE at the site level showed that this syndrome was associated with large tonnage and rapid production of rainbow trout for the table market. The data collected during this study enabled the identification of those sites that were most likely to present with RTGE the following year and this information was used to study the epidemiology of RTGE at the unit level. A prospective longitudinal study was undertaken in 12 RTGE-affected UK sites. It described in detail the impact, presentation, current control strategies and spread pattern of RTGE within affected UK sites. The risk factors associated with RTGE presence and severity were also investigated. Data were collected for each productive unit (i.e. cage, pond, raceway or tank) on the mortalities, fish origin, site management and environmental factors. RTGE was identified using a case definition based on gross pathological lesions. Analysis of these data revealed that RTGE behaved in an infectious manner. This conclusion was supported by the presence of a pattern typical of a propagating epidemic within affected units. Also, the risk of an unaffected unit becoming RTGE positive was increased if it had received fish from or was contiguous to a RTGE-affected unit. The presentation also suggested an incubation period of 20-25 days. Risk factor analysis identified management and environmental risk factors for RTGE, including high feed input and stressful events, which could be used to generate a list of control strategies. A study of the histopathological and ultrastructural presentation of RTGE was conducted. The location of segmented filamentous bacteria (SFB) and pathological changes found in affected fish were examined. Pyloric caeca were the digestive organ where SFB were found more frequently and in higher numbers, suggesting that this was the best location to detect SFB in RTGE-affected trout. Scanning and transmission electron microscopy revealed a previously undescribed interaction of SFB with the mucosa of distal intestine and pyloric caeca and this included the presence of attachment sites and SFB engulfment by enterocytes, as previously described in other host species. The SFB were not always adjacent to the pathological changes observed in the digestive tract of RTGE-affected trout. Such changes included cytoskeletal damage and osmotic imbalance of enterocytes, with frequent detachment. These observations suggested that if SFB are indeed the cause of RTGE their pathogenesis must involve the production of extracellular products. Analysis of the gross presentation and blood biochemistry in RTGE-affected fish was used to examine the patho-physiologic mechanisms of RTGE. To enable identification of positive RTGE cases for this study, a case definition was created from the information available on RTGE gross presentation in the literature. This case definition was assessed in a sample including 152 fish cases and 152 fish controls from 11 RTGE-affected UK sites, matched by unit of origin. The analysis of these fish using bacteriology, packed cell volume (PCV) and histopathology revealed that RTGE occurred simultaneously with other parasitic and bacterial diseases in a percentage of fish identified with this case definition. With the information gained after analysing the gross presentation, RTGE-affected fish without concurrent disease were selected for the study of the pathogenesis, which included blood biochemical analyses. These analyses revealed a severe osmotic imbalance, and a reduced albumin/globulin ratio suggesting selective loss of albumin, typical for a protein losing enteropathy. The role of the SFB “Candidatus arthromitus” in the aetiology of RTGE was assessed using a newly developed “C. arthromitus”-specific polymerase chain reaction assay (PCR) in conjunction with histological detection. This technique was applied to eight different groups of trout, including an RTGE-affected group and seven negative control groups. This analysis was conducted on DNA extracted from paraffin wax-embedded tissues as well as fresh intestinal contents. The results revealed the presence of “C. arthromitus” DNA in apparently healthy fish from sites where RTGE had never been reported. Additionally, SFB were observed histologically in two trout from an RTGE-free hatchery. These findings do not permit the exclusion of “C. arthromitus” as the aetiological agent for RTGE, although they suggest that the presence of these organisms in the digestive system of healthy trout is not sufficient to cause clinical disease, and therefore other factors are necessary. In conclusion, this study has used a multidisciplinary approach to the study of RTGE which has generated scientific information related to the epidemiology, pathogenesis and aetiology of this syndrome. The results of this project have suggested priority areas where further work is required, including experimental transmission of RTGE, field assessment of the control strategies proposed and further investigation into the aetiology of RTGE.

Les anatomopathologistes disposent d’outils permettant d’assister leurs décisions cliniques et d’évaluer les risques de récidive des patientes atteintes d’un cancer du sein. Parmi ceux-ci, le grade histologique du cancer du sein divise les patientes en trois sous-groupes pour lesquels le grade histologique 1 et 3 sont respectivement associés à de bons et mauvais pronostics. Cependant, cet outil est loin d’être parfait, dû au manque de reproductibilité de ce système et du risque de récurrence intermédiaire, peu informatif, des patients classés dans la catégorie « grade 2 ».

Afin de mieux caractériser ces tumeurs de risque intermédiaire, notre laboratoire a introduit un score appelé « Gene expression Grade Index (GGI) », basé sur l’expression de 97 gènes définis par microarrays. De façon intéressante, ce GGI permet de diviser les patientes de grade histologique 2, sur base de leur profil d’expression, en 2 groupes correspondant aux tumeurs de grade 1 ou aux tumeurs de grade 3. Cependant, bien que le GGI apporte une information importante, son applicabilité clinique est limitée par son prix et la nécessité d’utiliser du matériel congelé.

Durant ce travail de thèse, nous avons transposé la signature microarrays en un test RT-PCR, appelé PCR-GGI, basé sur l’expression de 8 gènes qui permet de reproduire les performances du GGI à partir de tissus congelés ou conservés dans de la paraffine. Cette amélioration permet de faciliter son utilisation en routine clinique.

De plus, nous avons approfondi notre connaissance du grade histologique, au niveau génomique et transcriptomique, et montré que les tumeurs mammaires (ER-positives) peuvent être divisées en deux groupes :un premier groupe de faible instabilité génomique, exprimant faiblement les gènes de prolifération et présentant un faible risque de récurrence ;et un deuxième groupe de haute instabilité génomique (impliquant principalement des amplifications localisées dans les régions 8q et 20q), une expression importante de gènes de prolifération et un mauvais pronostic.

D’autre part, les carcinomes canalaires in situ (DCIS) présentant des similarités avec les tumeurs invasives, nous avons voulu mieux comprendre le comportement du grade tumoral parmi ces tumeurs pré-invasives. Nous avons donc intégré le PCR-GGI au VNPI et défini le VNPI-GGI. Comparé au VNPI classique, le VNPI-GGI identifie mieux les patientes qui vont récidiver tôt dans les groupes de risque intermédiaire et haut, et permet donc d’éviter le sur-traitement.

Cependant, le calcul du VNPI est un travail fastidieux et le PCR-GGI seul ne permet pas de prédire les risques de récidives des DCIS. Nous avons donc cherché un nouveau marqueur pronostique. Alors, qu’il existe des preuves de plus en plus nombreuses supportant l’importance du rôle anti-tumoral des cellules myoépithéliales, nous avons montré qu’une diminution de l’expression de CD10 – un marqueur des cellules myoépithéliale – était hautement corrélée au risque de récidive. Ces résultats soulignent l’importance tant de l’agressivité de la tumeur que de son environnement directe, dans la progression tumorale.

En terme d’applications, les résultats obtenus durant ce travail de thèse nous ont permis de développer des outils utilisables par les cliniciens afin d’améliorer la prise en charge des patientes.

Traditional histopathological tools routinely used to evaluate breast cancer prognosis are designed to assist physicians in their evaluation of clinical outcome. The histological grade of invasive breast cancer, that assigns patients to one of 3 groups for which histological grade 1 and 3 tumors are respectively associated with lower and higher rate of recurrence, has long provided clinically important prognostic information. However, this tool is far from perfect due to concern over reproducibility and intermediate risk of recurrence of the histological grade 2 that is not informative for clinical decision.

To better characterize tumors classified as histological grade 2, our group has introduced a score called Gene expression Grade Index (GGI) based on a cassette of 97 genes defined by Microarrays. Interestingly, the GGI was able to reclassify patients with histological grade 2 tumors into 2 groups with distinct clinical outcomes similar to those of histological grade 1 and 3, respectively. However, its clinical applicability still remains expensive and often requires frozen tissue.

During this thesis work, we have transposed the GGI onto a qRT-PCR assay, called PCR-GGI, based on a set of 8 genes that could recapitulate in an accurate and reproducible manner the prognostic performance of GGI using both frozen and paraffin-embedded (FFPE) tumor samples, to facilitate its use in clinical practice.

Moreover, we have explored histological grade of invasive breast cancer at genomic and transcriptomic level and we have shown that two classes of ER-positive invasive breast cancer are observed: a first of low genomic instability, low proliferation gene expression and low risk of recurrence; and a second of high genomic instability (implying a major role for amplification of region located on chromosome arms 8q and 20q), high proliferation gene expression and worse prognosis.

In addition, since Ductal Carcinoma in situ (DCIS) and invasive breast cancer show concordant biologic behavior, we attempted to better understand the molecular basis of grade in pre-invasive breast cancer. We have then incorporated the PCR-GGI in the VNPI and defined the VNPI-GGI to improve its prognostic value. Compared to the classic VNPI, the VNPI-GGI had a better potential to identify early relapsing patients in the intermediate and high score group, and avoid under treatment in high-risk DCIS patients.

However, VNPI scoring is a tedious work and PCR-GGI alone can’t predict recurrence in pre-invasive breast cancer. We aimed then to find news prognosis marker in the field of DCIS. As there is now growing body of evidence supporting the role of myoepithelial cells (MECs) as natural tumor suppressors, we have showed that a decrease of CD10 expression- a surface biomarker of MECs – was significantly associated with an increased risk of relapse.

These results highlight the importance of assessing intrinsic DCIS properties as well as juxta-tumoral stroma, both seems to have a major role in DCIS progression.

In terms of applications, from these results obtained during this thesis work, we developed methods applicable into clinical practice to improve patients management.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Els nucleòtids extracel·lulars, com l’ATP, i els nucleòsids que deriven de la seva hidròlisi, com l’adenosina, estan àmpliament distribuïts en tots els òrgans i sistemes animals i estan considerats com a potents molècules senyalitzadores que, a través de receptors purinèrgics o purinoceptors (de tipus P1 i P2), intervenen en diverses funcions fisiològiques i fisiopatològiques de l’aparell reproductor. La concentració extracel·lular de nucleòtids i nucleòsids ve regulada per enzims de membrana anomenats ecto-nucleotidases, els quals hidrolitzen seqüencialment l’ATP fins a adenosina, controlant així, els seus nivells extracel·lulars i en conseqüència les vies de senyalització derivades de la unió d’aquests lligands als purinoceptors. Existeixen quatre famílies d’ecto-nucleotidases: a) la família de les ecto­nucleòsid trifosfat difosfohidrolases (E-NTPDases) o família CD39 consta de vuit membres (NTPDasa 1­8) quatre dels quals s’expressen a la membrana plasmàtica (NTPDasa 1, 2, 3 i 8) i hidrolitzen amb diferents afinitats l’ATP i l’ADP fins a AMP, b) la família de les ecto-nucleòtid pirofosfatases/fosfodiesterases (-E-NPPs) conté set membres (NPP 1-7) dels quals tres (NPP 1, 2 i 3) hidrolitzen l’ATP i l’ADP fins AMP, c) les fosfatases alcalines, catalitzen la degradació de nucleòtids tri­, di-i monofosfats indistintament, i d) la família de les 5’-nucleotidases o CD73 com a únic membre de membrana que actua extracel·lularment hidrolitzant l’AMP fins adenosina. S’entén per senyalització purinèrgica el conjunt d’efectes biològics (autocrins i paracrins) produïts per les purines i les pirimidines en actuar com a lligands extracel·lulars. I, al complex molecular implicat en l’activació, la conservació i la terminació de la senyalització purinèrgica se li ha donat el nom de Purinoma, i inclou tant els lligands, com els receptors, els transportadors /canals i els enzims de degradació. Estudis previs del nostre grup demostren expressió d’algunes de les ecto-nucleotidases més importants al sistema reproductor masculí. No es coneixia encara, però, el paper de les ecto-nucleotidases al sistema reproductor femení ni en les patologies relacionades amb aquest aparell com és el càncer d’endometri, aspectes que han estat estudiats en aquesta tesi doctoral. Així, a través de tècniques histològiques, immunològiques i moleculars, hem estudiat l’expressió gènica i proteica així com l’activitat de les ecto­nucleotidases a l’aparell reproductor femení, en el endometri funcional i atròfic humà, i en el càncer d’endometri. A més, hem posat a punt dos models cel·lulars (línies cel·lulars d’adenocarcinoma endometrial humà i cultius primaris de cèl·lules de l’estroma endometrial humà) on estudiar la regulació de les ecto-nucleotidases. Els nostres resultats mostren expressió i activitat d’aquests enzims a l’endometri amb marcades diferències al llarg del cicle i després de la menopausa, indicant una possible regulació hormonal d’aquestes. També hem detectat una elevada expressió de dues de les ecto-nucleotidases que controlen directament els nivells extracel·lulars d’adenosina, CD39 i CD73, en el carcinoma endometrial Tipus I i Tipus II evidenciant així el seu potencial us com a dianes terapèutiques en oncologia. Per tot això, proposem les ecto-nucleotidases com a possibles biomarcadors de fertilitat i amb utilitat pronòstica i diagnòstica de l’adenocarcinoma endometrial.
Extracellular ATP and its hydrolysis product adenosine, acting through specific receptors collectively named purinergic receptors, modulate various reproductive functions. There are four major groups of ecto-nucleotidases that control the levels of extracellular ATP and adenosine and thus their availability at purinergic receptors: a) ecto-nucleoside triphosphate diphosphohydrolases, b) ecto-nucleotide pyrophosphatase/phosphodiesterase, c) ecto-5’­nucleotidase, and d) alkaline phosphatase. The Purinome is the molecular complex implicated in the triggering, maintenance and termination of the purinergic signalling which is the group of biological effects (autocrine or paracrine) produced by the purines and pyrimidines acting as a extracellular ligand and it is form by the ligands, receptors, transporters/channels and degrading enzymes. Previous studies of our group demonstrate expression of some of the most important ecto-nucleotidases in the male reproductive tract, but the role of the ecto-nucleotidases in the female reproductive system in health and disease were unknown. In this thesis, we have studied both aspects. We studied the gen and protein expression and the enzyme histochemistry of the main ecto-nucleotidases in the female reproductive tract, in the human cyclic (proliferative and secretory) and atrophic endometria, and in endometrial cancer. In addition, we have set up two cellular models (human endometrial adenocarcinoma cell lines and primary cultures of endometrial stromal cells) to study the regulation of the ecto-nucleotidases. Our results show expression and activity of these enzymes in the endometrium with marked changes along the cycle and after the menopause, indicating a hormonal regulation. Also we have detected an elevated expression of two of the ecto-nucleotidases that directly control the adenosine extracellular levels (CD39 and CD73), in the endometrial cancer type I and type II. Our results support the growing body of evidence that CD39 is a potential therapeutic target for cancer immunotherapy. And also reinforce the relevance of CD73 in tumors. We suggest that the ecto­nucleotidases can be used as a biological markers of fertility with prognostic and diagnostic utility in endometrial cancer.

O significado prognóstico do fenômeno de regressão espontânea em melanoma, especialmente nas lesões finas, tem sido controverso. Estudos recentes sugerem que regressão extensa e tardia (fibrótica) possa estar relacionada a pior prognóstico. Linfangiogênese e angiogênese predizem metástase em melanoma. Objetivos: Analisar a densidade microvascular linfática e panvascular em melanomas extensivo-superficiais finos (Breslow 1,0 mm), comparando: melanomas regressivos e não-regressivos, área regressiva e não-regressiva de um mesmo tumor , os diferentes estágios de regressão (precoce ou inflamatória e tardia) de um mesmo tumor e correlacionar angiogênese e linfangiogênese a fase de crescimento tumoral. Métodos: Análise retrospectiva, histopatológica e estudo imunoistoquímico de melanomas com os anticorpos monoclonais D2-40 (37 tumores, sendo 16 regressivos e 21 não-regressivos como controles) e CD31 (29 tumores, sendo 13 regressivos e 16 não regressivos como controles), e posterior quantificação da densidade microvascular por análise de imagem. Resultados: Foi encontrada maior densidade microvascular linfática na fase tardia de regressão quando comparada à área dos melanomas regressivos que não apresentava regressão (controle interno). Conclusões: A fase tardia da regressão espontânea nos melanomas finos apresentou maior densidade microvascular linfática, fato que pode relacioná-la a pior prognóstico, já que densidade microvascular linfática estaria relacionada a risco aumentado de disseminação metastática. Essa suposição necessita ser confirmada por um maior seguimento dos nossos casos para detecção de metástases linfonodais
The prognostic significance of spontaneous regression in melanoma, especially thin lesions, has been a controversial issue. Recent studies suggest that extensive and late regression may be related to worse prognosis. Angiogenesis and lymphangiogenesis predict metastatic spread in melanoma. Objectives: To quantify lymphatic and panvascular microvascular density in thin (Breslow 1,0 mm) superficial spreading melanomas comparing: regressive and non-regressive melanomas, regressive and non-regressive areas from the same tumor; early and late histological stages of regression in the same tumor and to correlate angiogenesis and lymphangiogenesis and tumor growth phase. Methods: We conducted retrospective study, histological examinations and immunohistochemical analyses using the monoclonal antibodies D2-40 (37 melanomas, 16 regressive and 21 non-regressive as controls) and CD31 (29 melanomas, 13 regressive and 16 non-regressive as controls) with subsequent quantification of microvascular lymphatic and panvascular density by image analysis. Results: We found greater lymphatic microvascular density in the late stage of regression compared with non-regressive area (internal control) of regressive melanomas. Conclusions: The late stage of spontaneous regression in thin melanomas showed greater lymphatic microvascular density and this may be related to worse prognosis as lymphatic microvascular density is related with increased risk of metastatic spread. This supposition must be confirmed by a longer follow-up for detection of lymph node metastases

Дисертація присвячена вивченню морфогенеза просторової організації міокарду у представників різних класів хребетних. Досліджені гістоморфологічні особливості архітектури тканинних компонентів міокарду на етапах кардіогенезу у Rana temporaria, курки, щура та людини. Визначені основні кроки структурних трансформацій елементів міокарду в аспекті їх взаємної орієнтації. Встановлена гістоархітектурна характеристика м’язового, гемомікроциркуляторного та сполучнотканинного компонентів з точки зору просторової орієнтації клітин та клітинних груп. Вивчені процеси клітинної проліферації та диференціювання у ранньому серці людини за допомогою імуногістохімічних маркерів Кі-67, CD-34, віментину та визначені особливості розподілення маркер-позитивних клітин. Створені тривимірні моделі ділянок стінки серця на різних етапах кардіогенезу вивчених об’єктів.

Thesis (Ph.D.)--University of the Witwatersrand, Faculty of Health Sciences, 2012
Background and Purposes Suicide by hanging is a relatively common occurrence. The actual cause of death in suicidal hanging is, however, controversial, having been attributed variously to asphyxia, carotid artery compression and vagal nerve stimulation. The aim of this Ph.D thesis was to determine the possible neurovascular cause of death in suicides by hanging by careful study of the anatomy and physiology of the neck region in relation to the ensuing pathology. The study was, therefore, approached from an anatomical, physiological, histological and pathological pespective. It therefore comprised a detailed exploration of the anatomy and physiology of the neck structures to match these with the underlying traumatised neurovascular structures, the latter trauma being brought about by the suicidal hanging process. Methods The methods used in the study included an investigation of the ligature and position of the ligature in relation to the level of the neck and the physical effects of the ligature on the skin and underlying anatomical structures. A careful and detailed dissection of the neck was undertaken and samples of the vessels and nerves were processed for histological study. Fifty consecutive cases of suicidal hanging and five “non-hanging” cases which served as controls were used in the study. In addition, ten cases of suicidal hanging not included in the study were subjected to occlusion studies by means of probe exploration. This technique and procedure was not carried out or applied to the cases included in the study for fear that the probe itself might produce artefactual damage to the delicate endothelium lining the inner layer of the vessel wall. The study was classified into various components such as: 1. Examination of the type and structure of the ligature material; 2. The position of the ligature on the neck, i.e. whether involving upper, middle, or lower third of neck and to correlate this position with the underlying anatomical structures subjected to the accompanying tensile, compressive and haemodynamic forces; 3. The physical effects of the ligature upon the skin and the underlying deeper neurovascular structures of the neck; 4. Meticulous “bloodless” dissection of the neck structures to corroborate any pathology noted with the above three criteria. Currently, all putative causes of death remain speculative; 5. Particular attention was paid to those structures most vulnerable to the compressive forces, tensile forces and haemodynamic forces operative in hanging. These comprise the neurovascular structures contained within the fibrous carotid sheath and the phrenic nerves in the neck, in particular with regard to the anatomical relationship of these structures to the positioning of the ligature. As far as analysing the forces involved, the engineering principles pertaining to these were interpreted in consultation with the Faculty of Engineering at the University of the Witwatersrand. Results The main findings of the study showed damage to vascular, neural (including phrenic nerve), carotid bodies and accessory glomal bodies. The vascular findings emerged following an examination of the total number of arteries in the study, namely, 300, the figure derived as follows: six arteries in each of the fifty hanging subjects, viz., the left common carotid artery, the right common carotid artery, the left internal carotid artery, the right internal carotid artery, the left external carotid artery and the right external carotid artery (6 x 50 = 300). The damage shown was particularly the case with regard to the finding of tears in the various layers of the vessel wall. These extended from the intima through to the adventitia or outermost layer of the vessel wall and these were further subdivided into being either single or multiple. The tears found ranged from those involving the intima alone (single tears being found in 17 (5.6%) of the 300 arteries examined and multiple tears in 37 (12.3%) of the 300 arteries examined., the intima extending to the internal elastic lamina (single tears being found in 20 (6.6%) of the 300 arteries examined and multiple tears in 8 (2.6%) of the 300 arteries examined), tears involving the intima and extending through to involve the media, i.e. intimomedial tears and whether these latter tears involved the inner-, middle-, or outer-thirds of the media (single or multiple). Single intimo-medial tears extending through the intima to involve the inner-third of the media comprised 6 (2.0%) of the arteries examined, those extending from the intima to involve the middle-third of the media comprised 3 (1.0%) of the 300 arteries examined and single intimo-medial tears extending through the intima to involve the outeriv third of the media similarly comprised 3 (1%) of the arteries examined. Multiple intimo-medial tears extending through from intima to inner-, middle-, and outerthirds of the media respectively, comprised 3 (1.0%), 5 (1.6%) and 1 (0.3%) of the arteries examined. Single tears involving the inner-third of the media alone comrised 6 (2.0%) of the 300 arteries examined, single tears involving the middle-third of the media comprised 9 (3.0%) of the arteries examined and single tears involving the outer-third of the media alone comprised 8 (2.6%) of the arteries examined. Multiple tears involving the inner-, middle and outer-thirds of the media respectively comprised 6 (2.0%), 13 (4.3%) and 16 (5.3%) of the arteries examined. Single tears involving both adventitia and media, i.e. adventitio-medial tears extending through the inner-, middle-, or outer-thirds of the media to involve the adventitia comprised 1 (0.3%), 2 (0.6%) and 6 (2.0%) respectively of the 300 arteries examined. Multiple adventitio-medial tears of the inner-, middle-, and outer-thirds of the media, respectively, comprised 0 (0.0%), 3 (1.0%) and 2 (0.6%) of the 300 arteries examined. Single tears of the adventitia alone comprised 21 (7.0%) of the arteries examined while multiple tears comprised 7 (2.3%). Complete circumferential transverse rupture of the vessel wall was found in 3 (1.0%) of the arteries examined while adventitial haemorrhage was found in 103 (34.3%) of the 300 arteries examined. The vascular findings were represented numerically in tabular form in the 50 hanging subjects in Table III and were further analysed and compared with regard to either unilateral or bilateral vessel involvement in the fifty (50) suicidal hanging subjects and the findings represented in Tables IIIa (unilateral involvement) and IIIb (bilateral involvement). Additional vascular findings comprised endothelial elevation/avulsion, internal elastic lamina dehiscence, subendothelial clefts, multiple medial fenestrations, adventitio-medial separation, vascular congestion and a vascular plane of cleavage. These were similarly represented in Table IV and analysed with regard to unilateral or bilateral involvement in Tables IVa and IVb. Endothelial elevation/avulsion was found in 295 (98.3%) of the 300 arteries examined, internal elastic lamina dehiscence in 290 (96.6%) of the arteries examined, subendothelial clefts in 289 (96.3%) of the arteries examined, multiple medial fenestrations in 17 (5.6%) of the arteries examined, adventitiomedial separation in 273 (91.0%) of the arteries examined, vascular congestion in 224 (74.6%) of the arteries examined and a vascular plane of cleavage in 98 (32.6%) of the arteries examined. These findings, unexpected, showed the extreme fragility and vulnerability of the intima and adventitia to the compressive and tensile forces acting on the vessel wall during hanging, being explicable not only on the basis of the various complex forces interacting simultaneously during hanging but on the magnitude of forces applied. A mathematical analysis, found at the end of the Discussion chapter, conducted in order to estimate the minimum peak pressure applied and exerted on the vessel wall during hanging, in collaboration with the School of Mechanical, Industrial and Aeronautical Engineering at the University of the Witwatersrand, confirmed the magnitude of these forces. The neural findings (Table V) were divided into neural congestion, neural haemorrhage, neural internal dehiscence, neural tearing and perineural separation and these were similarly analysed with regard to either unilateral or bilateral involvement in the fifty hanging subjects (Tables Va and Vb). Neural congestion was found in association with 20 (6.6%) of the 300 arteries examined, neural haemorrhage in14 (4.6%), neural internal dehiscence in 54 (18.0%), neural tearing in 35 (11.6%) and perineural separation in 112 (37.3%). Neural ganglionic findings were similarly divided into ganglionic congestion, ganglionic haemorrhage, ganglionic internal dehiscence and ganglionic tearing. Ganglionic congestion, in association with the 300 arteries examined, was found in 20 (6.6%), ganglionic haemorrhage in 8 (2.6%), ganglionic internal dehiscence in 15 (5.0%) and ganglionic tearing in 6 (2.0%). The findings in the carotid bodies were divided into carotid body congestion, carotid body haemorrhage, carotid body internal dehiscence and carotid body tearing. Carotid body congestion, in association with the 300 arteries examined, was found in 8 (2.6%), carotid body haemorrhage in 2 (0.6%), carotid body internal dehiscence in 4 (1.3%) and carotid body tearing in 2 (0.6%). Accessory glomal body findings were, once again, divided into accessory glomal congestion, accessory glomal haemorrhage, accessory glomal internal dehiscence and accessory glomal tearing. However, in view of the close anatomical association between the accessory glomal bodies and the adventitia of the arterial walls, an additional pathological finding of accessory glomal adventitial separation emerged. Accessory glomal congestion, in association with the 300 arteries examined, was found in 20 (6.6%), accessory glomal haemorrhage in 7 (2.3%), accessory glomal internal dehiscence in 50 (16.6%), accessory glomal tearing in 18 (6.0%) and accessory glomal adventitial separation in 124 (41.3%). This latter finding once again demonstrated the vulnerability of the adventitial layer of the vessel wall to tensile forces, separating it from its associated structures. Damage to the phrenic nerves and surrounding muscles, underlying the site of ligature application, was similarly found, suggesting a role for phrenic nerve stimulation with consequent diaphragmatic paralysis in contributing to death in the hanging process. Discussion and Conclusion In this Ph.D thesis the principles of dimensional analysis i.e., the breaking down of a complex phenomenon into its component parts, have been applied. However, in view of the complexity and proximity of structures to one another in the neck, consisting not only of the rigid hyoid-larynx complex and vertebral column but also the integrated vascular and neural structures, it appears that not one single biological mechanism can be ascribed and attributed to the cause of death in suicidal hanging. Rather, it appears that unconsciousness and death causation appears to be multifactorial. Both the sympathetic and parasympathetic arms of the autonomic nervous system are involved, often with antagonistic and therefore paradoxical effects. In a ddition, pressure to the phrenic nerve, not previously considered in playing a role in death causation in hanging, may, it is suggested, be a major contributory factor in death causation. This nerve, the innervation to the major muscle of respiration, i.e. the diaphragm, in a neural response to the compressive and tensile forces in hanging, fixes the diaphragm in a state of inspiratory paralysis. This latter effect would be further augmented by neural stimulation of the accessory muscles of respiration, i.e. the sternocleidomastoid and scaleni muscles, similarly lying deep to the site of ligature application, contributing to the thoracic cage becoming fixed in a state of inspiratory paralysis. This latter effect, as described in that section of the Discussion chapter dealing with an analysis of the physiological functions at play, is brought about by initiation of the dynamic and static stretch reflexes occurring in these muscles on application of a compressive or tensile stimulus. Compression of the carotid arteries, on the other hand, results, as shown, not only in major damage to these vessels and their accompanying veins, but, in addition, must produce a dramatic element of cerebral ischaemia with ensuing loss of consciousness. It thus appears that loss of consciousness is the critical factor for it is the state when the victim is no longer able to save himself or herself. Without loss of consciousness survival may occur, but with it, death becomes inevitable. The question then arises :- what is the cause of unconsciousness? In physiological terms, carotid artery occlusion induces rapid unconsciousness, i.e. within 11 to 12 seconds, resulting ultimately in death. In other words, the sudden application and unremitting pressure of the ligature must inevitably result in death. On the other hand, the sudden application of a ligature with consequent vagal nerve compression may produce instantaneous cardiac arrest with cessation of blood flow to the brain and resultant loss of consciousness. This event would produce unconsciousness in less than the time period of 11 seconds of carotid artery occlusion (although the brain continues to survive for several minutes thereafter despite cessation of heart beat). If, however, unconsciousness is contributed to by phrenic nerve compression, it would not be instantaneous as shown by the fact that one can normally hold one’s breath for several minutes (as underwater swimmers do) and unconsciousness does not supervene either instantaneously or within 11 seconds. In short, unconsciousness would not occur within 11 seconds in the case of compression of the phrenic nerve unless a more critical factor supervenes.Thus, the rapidity of onset of unconsciousness appears to be the critical factor in determining the progression to ultimate (and inevitable) death. Moreover, as pointed out in the Materials and Methods chapter, the carotid arteries in several tested cases would not allow the passage of a probe through the obstructed arteries beneath a tightly applied ligature. This obstruction would, therefore, appear to be the initiator of the deadly unconsciousness factor, although respiratory arrest would be compounded by neural and muscular factors. While in this thesis the principles of dimensional analysis i.e., the breaking down of a complex phenomenon into its component parts have been applied, the principles of integrated analysis i.e., the combining and synthesis of separate parts into a whole have also been attempted. In essence, while it is suggested that the neural elements play a pivotal role in the hanging process due to the neural effect on both brain and heart as a result of autonomic nervous system stimulation and the function of the phrenic nerve in respiration, it appears that multiple factors, acting in concert, simultaneously or in rapid sequence to one another, all play a role in contributing to death causation in the hanging process.

Purpose: To highlight the histopathological characteristics of the skin in congenital clubfoot and correlate the clinical findings in clubfoot with the changes in the dermal layers. Materials and methods: One hundred skin specimens, from 77 infants (6 to 12 months), were studied between 2004 and 2008. Using the Pirani scoring system, the clinical severity was recorded. The mobility of the skin and the correctability of the medial ray were assessed clinically. A skin specimen (1cm x 1mm) was taken from the medial side of the foot at surgery following failed plaster treatment. The layers were studied under light microscopy. The thickness of the dermis and the histopathological features of clubfoot skin were compared with 10 normal skin specimens. Results: The dermis of clubfoot skin showed significant fibrosis with thick bundles of collagen fibres (P = .001) on Haematoxylin and Eosin staining (H&E). The dermal thickness ranged between 1.0mm and 5.2mm in clubfoot skin, compared with controls (0.64-1.28mm). Fibrosis extended into the subcutis in a septolobular fashion in 95% of the cases. Significant atrophy of eccrine glands was seen in 98% (P = .001). Hair follicles were absent in 78%. The elastic fibres of clubfoot skin, stained with Elastic van Gieson staining (EVG), showed hypertrophy in varying degrees in all skin specimens. They were fragmented, with loss of their parallel arrangement. There was no significant inflammatory reaction in the dermis. The Pirani score was significantly increased (mean 7.8). Discussion: Fibrosis and thickening of the dermis were the most significant histopathological features of the clubfoot skin. The elastic fibres were also abnormal. There was atrophy of the skin appendages due to the fibrosis. There was a strong correlation between the Pirani score and the severity of the deformity(P 0.016). The cases with poor outcome had a higher score than those with a satisfactory outcome.Lack of a significant inflammatory reaction suggests that neither the serial manipulations of the foot, nor the repeated plaster cast changes, were responsible for the dermal fibrosis, which is probably present from birth and contributes to the deformity.
Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2012.

Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Filosofie a dějiny přírodních věd MUDr. Jan Hrudka Nádory v dějinném a kulturním kontextu v novověku Tumours in historical and social context in the modern period Disertační práce Školitel / Supervisor: Prof. RNDr. Stanislav Komárek, Dr. Praha, 2017 SUMMARY: The PhD thesis called Tumours in historical and social context in the modern period is an attempt to describe a change of medical thinking in modern period; science and medicine turns from antique humoral pathology, explaining all diseases as an imbalance of the four body humours, to pathological anatomy and experimental physiology. In the point of view of pathological anatomy, the viscera of diseased person are no more "screen" or "mirror" of the disease, but it becomes directly the "stage" or "theatre" of the acting disease. This shift in the thought may be labelled as movement from humoralism to localism or ontologism; the disease isn't just abnormal amount of some natural juice any more, but becomes new original entity. This change undergoes the understanding of tumours and cancerous disease as well. Instead of antique understanding tumours as precipitates of black bile, the cell theory occurs in the 19th century. This theory explains tumours as a mass of cells undergoing excessive...

Indiana University-Purdue University Indianapolis (IUPUI)
Candida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals. C. glabrata in particular has become a significant concern due to the increase in clinical isolates that demonstrate resistance to triazole antifungal drugs, the most prevalent treatment for such infections. Triazole drugs target the ERG11 gene product and prevent C-14 demethylation of the first sterol intermediate, lanosterol, preventing the production of the pathways end product ergosterol. Ergosterol is required by yeast for cell membrane fluidity and cell signaling. Furthermore, C. glabrata, and not C. albicans, has been reported to utilize cholesterol as a supplement for growth. Although drug resistance is known to be caused by an increase in expression of drug efflux pumps, we hypothesize a second mechanism: that the overuse of triazole drugs has lead to the increase of resistance by C. glabrata through a 2-step process: 1) the accumulation of ergosterol auxotrophic mutations and 2) mutants able to take up exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically. Two groups of sterol auxotrophic C. glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol precursor. Sterol auxotrophs have been created in C. glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol pathway to assess which ergosterol mutants will take up sterols aerobically. Random and site-directed mutagenesis was also completed in ERG25 of Saccharmoyces cerevisiae. The ERG25 gene encodes a sterol C-4 methyloxidase essential for sterol biosynthesis in plants, animals, and yeast. This gene functions in turn with ERG26, a sterol C-3 dehydrogenase, and ERG27, a sterol C-3 keto reductase, to remove two methyl groups at the C-4 position on the sterol A ring. In S. cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif. We have conducted site-directed and random mutagenesis in the S. cerevisiae wild-type strain SCY876. Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species. Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for site-directed mutagenesis. The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required for the oxidation-reduction in the C-4 demethylation reaction.