Made available in DSpace on 2015-04-11T13:38:22Z (GMT). No. of bitstreams: 1 francisca.pdf: 878093 bytes, checksum: 251097fdfe9003556935b61ec3a58ab8 (MD5) Previous issue date: 2012-06-27 / The human papillomavirus (HPV) is recognized as a major risk factor related to cervical cancer. The identification of HPV types of high risk can aid in the prevention of malignant lesions of the cervix. This study aims to determine and genotype HPV in women with a cytological result of ASC-US and LSIL. This is a cross-sectional study with an analytical component in women attended the Fundação Alfredo da Matta - FUAM. The patients were selected from the file examination of the Laboratory of Cytology of FUAM from January 2009 to July 2011 and requested to attend the DST clinic through active search in order to make a new collection of material for revaluation cytological, molecular detection and genotyping of HPV. Molecular detection was performed by Nested-PCR targeting the L1 region of the viral capsid. PCR products were analyzed on an agarose gel. Out of 100 patients selected, 70% (70/100) participated in the study, 34 of them had the cytological result of ASC-US and LSIL of 36. After reevaluation cytological 8 (11.4%) patients had cytology within normal limits, 33 (47.2%) inflammatory cytology, 22 (31.4%) ASC-US, 6 (8.6%) LSIL, 1 (1.4%) HSIL. HPV was identified in 28.6% (20/70) of the samples. Of the 20 patients HPV DNA-positive 1 had cytology within normal limits, 6 inflammatory cytology, 10 ASC-US and 2 LSIL and 1 HSIL. After the genotyping were identified the following HPV types 6, 16, 58, 61, 70, 83, 84 and 85. The most prevalent HPV 25% was HPV 58. The presence of HPV high-risk oncogenic stresses the importance of actions aimed at preventing the transmission of this virus and tracking of diseases in the city of Manaus. / O Papillomavírus humano (HPV) é reconhecido como o principal fator de risco relacionado do câncer cervical. A identificação de tipos de HPV de alto risco pode auxiliar na prevenção de lesões malignas do colo do útero. Este estudo tem como objetivo determinar e genotipar o HPV em mulheres com resultado citológico de Lesão Intraepitelial de Baixo grau (LSIL) e Células Escamosas Atípicas Significado Indeterminado (ASC-US). Trata-se de um estudo de corte transversal com componente analítico em mulheres atendidas na Fundação Alfredo da Matta - FUAM. As pacientes foram selecionadas a partir do arquivo de exames do Laboratório de Citologia da FUAM no período de janeiro de 2009 a julho de 2011 e solicitadas a comparecer ao ambulatório de DST por meio de busca ativa, a fim de fazer uma nova coleta de material para reavaliação citológica, detecção molecular e genotipagem do HPV. A detecção molecular foi realizada pela técnica de Nested-PCR tendo como alvo a região L1 do capsídeo viral. Os produtos da PCR foram analisados em gel de agarose. Do total de 100 pacientes selecionadas, 70% (70/100) participaram do estudo, sendo que 34 delas tinham resultado citológico de ASC-US e 36 de LSIL. Após reavaliação citológica 8(11,4%) pacientes apresentaram citologia dentro dos limites da normalidade, 33(47,2%) citologia inflamatória, 22(31,4) ASC-US, 6(8,6%) LSIL, 1(1,4%) HSIL. O HPV foi identificado em 28,6% (20/70) das amostras examinadas. Das 20 pacientes HPV-DNA positivas 1 caso apresentou citologia dentro do limite da normalidade, 6 citologia inflamatória, 10 ASC-US, 2 LSIL e 1 caso apresentou HSIL. Após a genotipagem foram identificados os seguintes tipos de HPV: 6, 16, 58, 61, 70, 83, 84 e 85. O HPV mais prevalente com 5 casos em 20 positivos foi o HPV 58. A presença do HPV de alto risco oncogênico destaca a importância de ações voltadas para a prevenção na transmissão desse vírus e no rastreamento das doenças relacionadas, na cidade de Manaus.

INTRODUÇÃO: A síndrome de Williams-Beuren (SWB; OMIM #194050) é causada por microdeleções em hemizigose de genes contíguos na região 7q11.23. Fácies típico, estenose aórtica supravalvar (EASV), deficiência intelectual, personalidade amigável e hiperacusia constituem as principais características da SWB. A deleção mais freqüente é de 1,55Mb, porém deleção de 1,84Mb também já foi descrita. Embora a técnica de hibridização in situ por fluorescência (FISH) ser amplamente utilizada na detecção da deleção, os marcadores microssatélites são considerados altamente informativos e de fácil manejo. OBJETIVOS: Testar os marcadores microssatélites para o diagnóstico da SWB, determinar o tamanho e origem parental da microdeleção, comparar as características clínicas entre os pacientes com diferentes tamanhos da deleção e origem parental. MÉTODOS: Foram estudados 97 pacientes com diagnóstico clínico de SWB utilizando cinco marcadores microssatélites: D7S1870, D7S489, D7S613, D7S2476 e D7S489_A. A partir do DNA genômico dos probandos e de seus genitores, foi realizada reação em cadeia da polimerase (PCR), seguida de eletroforese em gel de poliacrilamida desnaturante (uréia, com 7,5 M) e os resultados visualizados com coloração de prata. RESULTADOS E DISCUSSÃO: Com cinco marcadores juntos, o resultado foi informativo em todos os pacientes. O marcador mais informativo foi D7S1870 (78,4%), seguido por D7S613 (75,3%), D7S489 (70,1%) e D7S2476 (62,9%). A deleção foi encontrada em 84 (86,6%) pacientes e sua ausência em 13 (13,4%) pacientes. A deleção de 1,55 Mb foi observada em 76/84 pacientes (90,5%) e 1,84 Mb em 8/84 pacientes (9,5%). A origem parental da deleção foi materna em 44/84 pacientes (52,4%) e paterna em 40/84 pacientes (47,6%). Alterações oculares foram mais freqüentes em pacientes com deleção. Não houve diferença nos achados clínicos entre o tamanho ou a origem parental da deleção, exceto para EASV, presente em maior freqüência nos pacientes com deleção paterna. Os resultados por marcadores microssatélites foram concordantes com FISH positivos, no entanto, em dois pacientes com FISH negativos, a microdeleção foi detectada apenas pelos marcadores. CONCLUSÃO: O uso de cinco marcadores microssatélites selecionados foi informativo em todos os pacientes. Em dois casos, os marcadores foram mais eficientes que FISH, portanto pode ser considerado um método alternativo para o diagnóstico molecular da SWB / INTRODUCTION: Williams-Beuren syndrome (WBS; OMIM 194050) is caused by hemizygous contiguous gene microdeletions at 7q11.23. Typical facies, supravalvular aortic stenosis (SVAS), mental retardation, overfriendliness and hiperacusis comprise typical symptoms in WBS. The most common deletion is 1.55 Mb, however 1.84 Mb deletion also has been described. Although fluorescence in situ hybridization (FISH) is widely used in the detection of deletion, microsatellite markers are considered highly informative and easily manageable. PURPOSE: Test the microsatellite markers for the diagnosis of WBS, to determine the size and parental origin of microdeletion, compare the clinical characteristics between patients with different sizes of the deletion and parental origin. METHODS: We studied 97 patients with clinical diagnosis of WBS using five microsatellite markers: D7S1870, D7S489, D7S613, D7S2476 and D7S489_A. From the genomic DNA of probands and their parents was performed polymerase chain reaction (PCR), followed by electrophoresis on denaturing polyacrylamide gel (urea, 7.5 M) and the results visualized with silver staining. RESULTS AND DISCUSSION: Using five markers together, the result was informative in all patients. The most informative marker was D7S1870 (78.4%) followed by D7S613 (75.3%), D7S489 (70.1%) and D7S2476 (62.9%). The deletion was found in 84 (86.6%) patients and absent in 13 (13.4%) patients. The deletion of 1.55 Mb was observed in 76/84 patients (90.5%) and 1.84 Mb in 8 / 84 patients (9.5%). The parental origin was maternal in 44/84 patients (52.4%) and paternal in 40/84 patients (47.6%). Abnormalities ocular were more frequent in the patients with a deletion. There were no clinical differences in relation to either the size or parental origin of the deletion, except for SVAS, present in more frequency in the patients with paternal deletion. The results for microsatellite markers were concordant with FISH positive, however, in two patients with negative FISH, the microdeletion was detected only by the markers. CONCLUSION: Using these five selected microsatellite markers was informative in all patients. In two cases, the markers were more efficient than FISH, thus can be considered an alternative method for molecular diagnosis in SWB

Magister Scientiae - MSc / Initially, a pilot study was carried out in order to reconstruct the major paternal and maternal lineages of the Muslim population living in the Cape metropolitan area. The Study has shown the ability of molecular genetic tools to give insight into the origins and history of local communities. The study was also used as a point of reference for the Strand Muslim Community project. Genetic variations of the Y-chromosome and mitochondrial DNA for the pilot study were analyzed using the RFLP technique. The SNaPshot mini-sequencing technique was used to genotype single nucleotide polymorphisms (SNP) on the Y-chromosome and mitochondrial DNA in 115 males from the Strand Muslim community. / South Africa

Microfabricated thermal cyclers for nucleic acid amplification by using polymerase chain reaction (PCR) have been demonstrated by several groups over the last decade, with improved cycling speed and smaller volumes when compared to conventional bench-top cyclers. However, high fabrication costs coupled with difficulties in temperature sensing and control remain impediments to commercialization. In this study we have used a silicon-glass device that takes advantage of the high thermal conductivity of silicon but at the same time utilizes minimum number of fabrication steps to make it suitable for disposable applications. The thermal cycler is based on noncontact induction heating developed in this group. The microchip reaction kinetics is studied for the first time in-situ during PCR, using a real-time fluorescence block that is capable of data acquisition every 0.7 s from the microchip. The fluorescence information from SYBR green I dye is used to optimize microchip amplification reactions and confirm the product by melting curve analysis. We have also developed a novel non-contact temperature sensing technique using SYBR green fluorescence that can be used for miniaturized PCR devices. The thesis is organized into the following chapters. In chapter 1 we introduce the basic biology ideas that are required to understand DNA amplification. DNA based analysis requires amplification of low initial concentrations to above detectable limits using a technique known as polymerase chain reaction (PCR). In this process, the sample is cycled through three thermal steps for 3040 times to produce multiple copies of DNA. In microchip PCR, conventional polypropylene tubes using 2050 µL volume are replaced by miniaturized devices using ~1 µL sample volumes. The device response improves in terms of ramp rate and total analysis time due to the small volume and smart design of the materials. In this chapter we summarize some of the issues important for miniaturized PCR devices and compare them with commercial tube PCR systems. In chapter 2 we describe the induction heating technique that was developed by our group for miniaturized devices. Induction heating is a noncontact heating technique unlike resistive heating which has been commonly used for microchip PCR. Though resistive heating is very efficient in terms of heat transfer efficiency, it is not suitable for disposable devices and requires multi-step microfabrication. Other non-contact heating techniques such as hot air and IR heating require larger size arrangements that are not suitable for miniaturized devices. The heating was verified by using a thermocouple soldered at the back of the secondary plate that was also used for feedback to the comparator circuit for control. The simple on-off circuit was able to control within ±0.1 ◦C with heating and cooling ramp rates of 25 ◦C/s and 2.5 ◦C/s respectively. In this chapter, we also describe the design and fabrication of the silicon-glass microchip fabricated in our lab. We have used silicon-glass hybrid device for PCR in which glass with a 2 mm drilled hole is anodically bonded to an oxidized silicon surface. The hole formed the static reservoir for 3 µL volume of amplification solution. During PCR, the solution needs to be cycled to high temperature of ~95 ◦C. Hence it was necessary to seal the tiny droplet of liquid against evaporation at this temperature. The devices after being filled by sample were covered by 4 µL of mineral oil to serve as an evaporation barrier. It was easy to recover the whole sample after amplification for further testing. Chapter 3 describes the development of a fluorescent block for SYBR green I dye (SG) used for real-time monitoring of the amplification. The block contains a blue LED for excitation, a dichroic beamsplitter, and silicon photodiode along with filters and focusing optics. Signal levels being weak, we incorporated lock-in detection technique. A TTL at 190 Hz was used to pulse the excitation source and detect the emission at the same frequency using a commercial lock-in amplifier. The block was first characterized using a commercial thermal cycler and polypropylene tubes with different dilution of initial template copy number, and the results crosschecked with agarose gel electrophoresis. Performing continuous monitoring every 0.7s within cycles, we discovered interesting features during extension which have not been studied previously. During the constant temperature extension step, the fluorescence shows a rise and then saturates until the temperature is cycled to the next set point. We have confirmed the same behavior in single cycle extension control experiments and established its connection with polymerase extension activity. We were thus able to extract the activity rate for two different kinds of polymerase in-situ during PCR. By monitoring PCR reactions with different fixed extension times, we were able to determine the optimum conditions for tube PCR. Chapter 4 implements the ideas of fluorescence monitoring from tube that was explained in the previous chapter for the silicon-glass microchip. Since the microchip uses parameters such as sample volume, ramp rates, stay time etc. which are different from tube PCR, we performed several initial test experiments to establish key capabilities such as low volume detection, 3 µL amplification, surface passivation of silicon-glass etc. The same fluorescence block was used to obtain DNA melting point information by continuously monitoring ds-DNA with SG while the temperature is ramped slowly (melting curve analysis). Depending on ds-DNA present, the fluorescence gives a melting temperature (TM ), which was used to calibrate the mix temperature with respect to the thermocouple sensor. After successfully calibrating the microchip, we confirmed complete chip PCR in silicon-glass devices using induction heater. The continuous monitoring of chip PCR gave similar curves as obtained previously for tubes except that the signal level was lower in silicon devices. Extension fluorescence information was used to find an optimum temperature for microchip that shows a maximum activity rate. Similarly the reaction time was optimized in-situ during PCR by using continuous fluorescence data in a feedback experiment. The commercial lock-in amplifier was also replaced by a homemade circuit to successfully pickup fluorescence signal from the microchip during melting curve analysis. In chapter 5, we describe a novel technique to sense the temperature from the microchip without touching the sample volume. Usually the temperature is monitored by a sensor spatially separated from the mix and it has always been challenging to measure the exact temperature accurately. Most of the sensors are not biocompatible and too large in size to be placed inside the small volume of liquid. We have developed a protocol that involves SG fluorescence with addition of excess sensor DNA to the amplification solution. The sensor DNA added into the mix is non specific to the primer used for amplification of the template. It therefore does not participate in the amplification and its number remains unchanged throughout the 3040 cycles of PCR. If the amount of sensor DNA is titrated accurately, it will saturate the fluorescence envelope which then shows very reproducible thermal response with cycling. We have used this thermal response of the fluorescence for feedback as a temperature sensor. The fluorescence feedback was shown to produce identical amount of product in comparison to thermocouple feedback. The product can also be verified by melting curve analysis if the sensor DNA is chosen carefully depending on the product. In this chapter we also discuss some preliminary experiments with smart devices that will use dye based temperature sensor and control along with fluorescence based amplification monitoring. Chapter 6 summarizes the thesis and discusses some of the future areas which can be explored in the field of microchip PCR devices.

<p>More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has&nbsp / been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term&nbsp / delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in&nbsp / Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother&rsquo / s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were&nbsp / collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of&nbsp / periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association&nbsp / between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant&nbsp / associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM&nbsp / against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to&nbsp / PLBW. </p>

Rickettsiae are obligate intracellular gram-negative bacteria transmitted by arthropod vectors. Rickettsiae sometimes cause disease in humans, typically with high fever, headache and occasionally an eschar. In Sweden, Rickettsia helvetica, belonging to the spotted fever group, is the only tick-transmitted rickettsia found free in nature. The pathogenic roll of R. helvetica has not been fully investigated, but it has been implicated in aneruptive fever and cardiac disease. This thesis describes parts of the transmission pathways of rickettsiae in Sweden. Rickettsia infection rates in ticks collected from birds were analysed, and the birds’ role as disseminators and reservoirs was studied. We found that more than one in ten ticks was infected with rickettsia bacteria, predominantly R. helvetica, and that migrating birds contribute not only to long-distance dispersion of bacteria, but also to an inflow of novel and potentially pathogenic rickettsia species, in this case R. monacensis and R. sp. strain Davousti-like species, into Sweden. Further, wild and domestic animals were found to have seroreactivity against R. helvetica, which shows that they are exposed and susceptible to rickettsia. Their role as reservoirs has not been determined, yet they may indirectly be involved in transmission of rickettsia to humans by infected ticks feeding on them. The seroreactivity in humans was also studied. Patients investigated for suspected Borrelioses and blood donors had detectable antibodies against Rickettsia spp., with the highest prevalence detected in the suspected Borreliosis group. This shows that humans in Sweden are exposed to and develop an immune response against rickettsia. The suspicion that R. helvetica may cause severe symptoms was verified by a patient with subacute meningitis where the bacterium was shown for the first time to cause an invasive infection with CNS involvement and where the bacterium was isolated from the patient’s cerebrospinal fluid. Growth characteristics and morphology of R. helvetica were studied to better understand invasiveness and virulence. The findings indicate that the invasiveness is comparable with other rickettsia, though R. helvetica seems to have a stable but slightly slower growth.  Rickettsia helvetica is endemic in Sweden and therefore needs to be considered when investigating disease after a tick bite. / Rickettsia är en liten, strikt intracellulär, gramnegativ bakterie som sprids med vektorer som fästingar, löss och loppor. Bakterien kan orsaka Rickettsios hos människa, en sjukdom där de vanligaste symtomen är hög feber, huvudvärk, muskelvärk och i vissa fall ett bettmärke (eschar). I Sverige är Rickettsia helvetica, som tillhör spotted fever gruppen (SFG), den enda fästingöverförda rickettsia bakterien som hittats allmänt i naturen. Patogeniciteten för R. helvetica är ofullständigt utredd, men ”aneruptive fever” och hjärtmuskelinflammation har rapporterats. Avhandlingen beskriver delar av smittkedjan för SFG rickettsia i Sverige. Bakteriernas förekomst i fästingar plockade från fåglar har studerats, likaså det ekologiska tryck som flyttfåglars bärarskap av infekterade fästingar bidrar med när de korsar olika världsdelar. Mer än var tionde fästing var infekterad med rickettsia bakterier, i huvudsak R. helvetica. Det visade sig att flyttfåglar bidrar inte bara till långväga spridning av bakterier utan även till införsel av nya potentiellt patogena rickettsiaarter, i detta fall identifierades R. monacensis och en R. sp strain Davousti liknande art. Vidare analyserades seroreaktivitet mot Rickettsia helvetica hos både tamdjur och vilda djur, vilket visade på antikroppsutveckling, som uttryck för smittexposition, i mer än vart femte djur. Djurens roll som reservoar för bakterien är inte klarlagd, men oavsett är djuren indirekt involverade i spridningen av bakterien till människa via infekterade fästingar som suger blod. Seroreaktivitet hos människa har också studerats. Patienter, provtagna på grund av misstanke om borreliainfektion, samt blodgivare hade detekterbara antikroppar mot Rickettsiae, med högst prevalens i gruppen med misstänkt borreliainfektion. Fynden visar att människor i Sverige är exponerade för och utvecklar en immunreaktion mot rickettsia. Att R. helvetica skulle kunna ge allvarlig sjukdom verifieras av ett patientfall med subakut meningit där bakterien för första gången visats ge invasiv infektion med påverkan på nervsystemet (CNS engagemang) och där bakterien isolerats från patientens ryggmärgsvätska.  Morfologi och tillväxtegenskaper för R. helvetica undersöktes för att bättre förstå bakteriens invasivitet och virulens. Fynden indikerar att invasiviteten är jämförbar med andra rickettsiaarter men R. helvetica verkar ha en stabil men något långsammare tillväxt. Rickettsia helvetica är endemisk i Sverige och måste tas i beaktande vid sjukdomsutredning efter ett fästingbett.

<p>More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has&nbsp / been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term&nbsp / delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in&nbsp / Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother&rsquo / s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were&nbsp / collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of&nbsp / periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association&nbsp / between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant&nbsp / associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM&nbsp / against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to&nbsp / PLBW. </p>

Philosophiae Doctor - PhD / More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother’s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to PLBW. / South Africa

Philosophiae Doctor - PhD / More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother’s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to PLBW.

The diploma thesis deals with micromethod of DNA isolation from ten differently processing food products containing pepper (Capsicum annum). PCR ready DNA was isolated by magnetic particles PGMA functionalized by carboxyl groups from homogenates prepared in lysis buffer with CTAB. Quantity and quality of DNA was estimated using spectrophotometric measurements and verified using PCR methods with primers specific for plant rDNA. Quality of isolated DNA varied depending on processing technology. DNA isolated from smoked grinded peppers and from heat treated food products was degraded and amplified with primers F_26S and R_26S (PCR product 220 bp) in contrary to the primers F_18S and R_5.8S (PCR product 700 bp). DNA isolated from the other food products was amplified with primers F_18S and R_5.8S (PCR product 700 bp). PCR product from one grinded pepper (Žitavská paprika) was cloned and sequenced.