Indiana University-Purdue University Indianapolis (IUPUI) / Short tandem repeat (STR) analysis is currently the most common method for processing biological forensic evidence. STRs are highly polymorphic and allow for a strong statistical power of discrimination when comparing deoxyribonucleic acid (DNA) samples. Since sample testing and court proceedings occur months, if not years apart, samples must be stored appropriately in the event additional testing is needed. There are generally accepted methods to store DNA extracts long-term; however, one universally recognized method does not exist. The goal of this project was to examine various methods of storage and make recommendations for a universal storage method that maintained DNA integrity over time. Four variables were evaluated: storage buffer, storage temperature, initial storage concentration and the effects of repeated freeze-thaw cycles. DNA quantity was assessed using real-time polymerase chain reaction and DNA quality was evaluated using STR genotyping. Overall, the Tris-EDTA (TE) buffer outperformed nuclease free water as a long-term storage buffer for DNA extracts. Stock tubes stabilized concentration better than single use aliquots when eluted with TE while tube type was not significant when water was the buffer. For samples stored in TE, temperature had no effect on DNA integrity over time, but samples stored in water were largely affected at room temperature. Additionally, the greater the initial DNA concentration, the less likely it was to degrade in water. As a result of this research, DNA extracts from forensic samples should be stored long-term in TE buffer with a minimum concentration of 0.1 ng/μL. When water is the buffer, frozen storage is recommended.

Komunikace předávaná skrze Internet zahrnuje komunikaci mezi pachateli těžké trestné činnosti. Státní zástupci schvalují cílené zákonné odposlechy zaměřené na podezřelé z páchání trestné činnosti. Zákonné odposlechy se v počítačových sítích potýkají s mnoha překážkami. Identifikátory obsažené v každém paketu jsou koncovým stanicím přidělovány po omezenou dobu, nebo si je koncové stanice dokonce samy generují a automaticky mění. Tato dizertační práce se zabývá identifikačními metodami v počítačových sítích se zaměřením na metody kompatibilní se zákonnými odposlechy. Zkoumané metody musejí okamžitě detekovat použití nového identifikátoru spadajícího pod některý z odposlechů. Systém pro zákonné odposlechy následně nastaví sondy pro odposlech komunikace. Tato práce se převážně zabývá dvěma zdroji identifikačních informací: sledováním mechanismu pro objevování sousedů a detekcí identity počítače na základě přesností měření času jednotlivých počítačů. V rámci dizertačního výzkumu vznikly grafy identit, které umožňují spojování identit s ohledem na znění povolení k odposlechu. Výsledky výzkumu je možné aplikovat v rámci zákonných odposlechů, síťové forenzní analýzy i ve vysokoúrovňových programově řízených sítích.

Indiana University-Purdue University Indianapolis (IUPUI) / Forensic DNA profiling is widely used as an identification tool for associating an individual with evidence of a crime. Analysis of a DNA sample involves observation of data in the form of an electropherogram, and subsequently annotating a DNA “profile” from an individual or from the evidence. The profile obtained from the evidence can be compared to reference profiles deposited in a national DNA database, which may include the potential contributor. Following a match, a random match probability is calculated to determine how common that genotype is in the population. This is the probability of obtaining that same DNA profile by sampling from a pool of unrelated individuals. Each state has adopted various laws requiring suspects and/or offenders to submit a DNA sample for the national database (such as California’s law that all who are arrested must provide a DNA sample). These profiles can then be associated with past unsolved crimes, and remain in the database to be searched in the event of future crimes. In the case of database samples, a physical sample of the offender’s DNA must be kept on file in the laboratory indefinitely so that in the event of a database hit, the sample is able to be retested. Current methods are to collect a buccal swab or blood sample, and store the DNA extracts under strict preservation conditions, i.e. cold storage, typically -20° C. With continually increasing number of samples submitted, a burden is placed on crime labs to store these DNA extracts. A solution was required to help control the costs of properly storing the samples. FTA™ paper was created to fulfill the need for inexpensive, low maintenance, long term storage of biological samples, which makes it ideal for use with convicted offender DNA samples. FTA™ paper is a commercially produced, chemically treated paper that allows DNA to be stored at room temperature for years with no costly storage facilities or conditions. Once a sample is required for DNA testing, a small disc is removed and is to be used directly in a PCR reaction. A high quality profile is important for comparing suspect profiles to unknown or database profiles. A single difference between a suspect and evidentiary sample can lead to exclusion. Unfortunately, the DNA profile results yielded from the direct addition have been unfavorable. Thus, most crime laboratories will extract the DNA from the disc, leading to additional time and cost to analyze a reference sample. Many of the profiles from the direct addition of an FTA™ disc result in poor quality profiles, likely due to an increase in PCR inhibitors and high concentrations of DNA. Currently, standardized protocols regarding the recommended locations for removal of a sample disc from a bloodspot on an FTA™ card does not exist. This study aims to validate the optimal location by comparing DNA profiles obtained from discs removed from the center, halfway, and edge locations of a bloodspot from 50 anonymous donors. Optimal punch location was first scored on the number of failed, partial or discordant profiles. Then, profile quality was determined based on peak characteristics of the resulting DNA profiles. The results for all three disc locations were 5.3% failed amplifications, 4.2% partial amplifications, and one case of a discordant profile. Profile quality for the majority of the samples showed a high incidence of stutter and the absence of non-template adenylation. Of the three disc locations, the edge of the blood stain was ideal, due to a presumably lower concentration of DNA and likely more dilute amount of the PCR inhibitor heme. Therefore, based on the results of this study, there is a greater probability of success using a sample from the edge of a blood stain spotted in FTA™ paper than any other location of the FTA™ card.

This study analyses how DNA evidence can be distorted by the behaviour of criminal investigators and role-players within the Criminal Justice System (CJS). This has a negative impact on justice resulting in further criminality. The study has resulted in revelatory weaknesses owing to constitutional violations which cause sound evidence to become futile as it will not be admissible in court. Justice is aborted. The researcher has further explained the properties of the pertinent terms, such as: mental illness, psycho-social functioning, DNA, forensic investigator, forensic psychology, and courts. Concepts are building blocks, hermeneutical distortion leads to the frustrating of what justice intends and this, in turn, leads to poor criminal investigation performance. It is submitted that not only ineptness, but also deception possibly evolves from genotypic to phenotypic type which causes unwelcome behaviour within the criminal justice system to surface. The frequency of monitoring psychological behaviour amongst criminal investigations is low, and it, therefore, also contributes to delict and the miscarriage of justice occurs. / Police Practice / M.A. (Criminal Justice)

There is a sharp increase in the number of old people living alone throughout the world. More often than not, such people require continuous and immediate care and attention in their everyday lives, hence the need for round the clock monitoring, albeit in a respectful, dignified and non-intrusive way. For example, continuous care is required when they become frail and less active, and immediate attention is required when they fall or remain in the same position for a long time. To this extent, various monitoring technologies have been developed, yet there are major improvements still to be realised. Current technologies include indoor positioning systems (IPSs) and health monitoring systems. The former relies on defined configurations of various sensors to capture a person's position within a given space in real-time. The functionality of the sensors varies depending on receiving appropriate data using WiFi, radio frequency identification (RFIO), ultrawide band (UWB), dead reckoning (OR), infrared indoor (IR), Bluetooth (BLE), acoustic signal, visible light detection, and sound signal monitoring. The systems use various algorithms to capture proximity, location detection, time of arrival, time difference of arrival angle, and received signal strength data. Health monitoring technologies capture important health data using accelerometers and gyroscope sensors. In some studies, audio fingerprinting has been used to detect indoor environment sound variation and have largely been based on recognising TV sound and songs. This has been achieved using various staging methods, including pre-processing, framing, windowing, time/frequency domain feature extraction, and post-processing. Time/frequency domain feature extraction tools used include Fourier Transforms (FTs}, Modified Discrete Cosine Transform (MDCT}, Principal Component Analysis (PCA), Mel-Frequency Cepstrum Coefficients (MFCCs), Constant Q Transform (CQT}, Local Energy centroid (LEC), and Wavelet transform. Artificial intelligence (Al) and probabilistic algorithms have also been used in IPSs to classify and predict different activities, with interesting applications in healthcare monitoring. Several tools have been applied in IPSs and audio fingerprinting. They include Radial Basis Kernel (RBF), Support Vector Machine (SVM), Decision Trees (DTs), Hidden Markov Models (HMMs), Na'ive Bayes (NB), Gaussian Mixture Modelling (GMM), Clustering algorithms, Artificial Neural Networks (ANNs), and Deep Learning (DL). Despite all these attempts, there is still a major gap for a completely non-intrusive system capable of monitoring what an elderly person living alone is doing, where and for how long, and providing a quick traffic-like risk score prompting, therefore immediate action or otherwise. In this thesis, a cost-effective and completely non-intrusive indoor positioning and activity-monitoring system for elderly people living alone has been developed, tested and validated in a typical residential living space. The proposed system works based on five phases: (1)Set-up phase that defines the typical activities of daily living (TADLs). (2)Configuration phase that optimises the implementation of the required sensors in exemplar flat No.1. (3)Learning phase whereby sounds and position data of the TADLs are collected and stored in a fingerprint reference data set. (4)Listening phase whereby real-time data is collected and compared against the reference data set to provide information as to what a person is doing, when, and for how long. (5)Alert phase whereby a health frailty score varying between O unwell to 10 healthy is generated in real-time. Two typical but different residential flats (referred to here are Flats No.1 and 2) are used in the study. The system is implemented in the bathroom, living room, and bedroom of flat No.1, which includes various floor types (carpet, tiles, laminate) to distinguish between various sounds generated upon walking on such floors. The data captured during the Learning Phase yields the reference data set and includes position and sound fingerprints. The latter is generated from tests of recording a specific TADL, thus providing time and frequency-based extracted features, frequency peak magnitude (FPM), Zero Crossing Rate (ZCR), and Root Mean Square Error (RMSE). The former is generated from distance measurement. The sampling rate of the recorded sound is 44.1kHz. Fast Fourier Transform (FFT) is applied on 0.1 seconds intervals of the recorded sound with minimisation of the spectral leakage using the Hamming window. The frequency peaks are detected from the spectrogram matrices to get the most appropriate FPM between the reference and sample data. The position detection of the monitored person is based on the distance between that captured from the learning and listening phases of the system in real-time. A typical furnished one-bedroom flat (flat No.2) is used to validate the system. The topologies and floorings of flats No.1 and No.2 are different. The validation is applied based on "happy" and "unusual" but typical behaviours. Happy ones include typical TADLs of a healthy elderly person living alone with a risk metric higher than 8. Unusual one's mimic acute or chronic activities (or lack thereof), for example, falling and remaining on the floor, or staying in bed for long periods, i.e., scenarios when an elderly person may be in a compromised situation which is detected by a sudden drop of the risk metric (lower than 4) in real-time. Machine learning classification algorithms are used to identify the location, activity, and time interval in real-time, with a promising early performance of 94% in detecting the right activity and the right room at the right time.

Virtual Private Networks (VPNs) is a booming business with significant margins once a solid user base has been established and big VPN providers are putting considerable amounts of money into marketing. However, there exists Website Fingerprinting (WF) attacks that are able to correctly predict which website a user is visiting based on web traffic even though it is going through a VPN tunnel. These attacks are fairly accurate when it comes to closed world scenarios but a problem is that these scenarios are still far away from capturing typical user behaviour.In this thesis, we explore and build tools that can collect VPN traffic from different sources. This traffic can then be combined into more realistic datasets that we evaluate the accuracy of WF attacks on. We hope that these datasets will help us and others better simulate more realistic scenarios.Over the course of the project we developed automation scripts and data processing tools using Bash and Python. Traffic was collected on a server provided by our university using a combination of containerisation, the scripts we developed, Unix tools and Wireshark. After some manual data cleaning we combined our captured traffic together with a provided dataset of web traffic and created a new dataset that we used in order to evaluate the accuracy of three WF attacks.By the end we had collected 1345 capture files of VPN traffic. All of the traffic were collected from the popular livestreaming website twitch.tv. Livestreaming channels were picked from the twitch.tv frontpage and we ended up with 245 unique channels in our dataset. Using our dataset we managed to decrease the accuracy of all three tested WF attacks from 90% down to 47% with a WF attack confidence threshold of0.0 and from 74% down to 17% with a confidence threshold of 0.99. Even though this is a significant decrease in accuracy it comes with a roughly tenfold increase in the number of captured packets for the WF attacker.Thesis artifacts are available at github.com/C-Sand/rds-collect. / Virtual Private Network (VPN) marknaden har växt kraftigt och det finns stora marginaler när en solid användarbas väl har etablerats. Stora VPN-leverantörer lägger dessutom avsevärda summor pengar på marknadsföring. Det finns dock WF-attacker som kan korrekt gissa vilken webbplats en användare besöker baserat på webbtrafik, även om den går genom en VPN-tunnel.Dessa attacker har rätt bra precision när det kommer till scenarier i sluten värld, men problemet är att dessa fortfarande är långt borta från att simulera typiskt användarbeteende.I det här examensarbetet utforskar och bygger vi verktyg som kan samla in VPNtrafik från olika källor. Trafiken kan användas för att kombineras till mera realistiska dataset och sedan användas för att utvärdera träffsäkerheten av WF-attacker. Vi hoppas att dessa dataset kommer att hjälpa oss och andra att bättre simulera verkliga scenarier.Under projektets gång utvecklade vi ett par automatiserings skript och verktyg för databearbetning med hjälp av Bash och Python. Trafik samlades in på en server från vårt universitet med en kombination av containeriseringen, skripten vi utvecklade, Unix-verktyg och Wireshark. Efter en del manuell datarensning kombinerade vi vår infångade trafik tillsammans med det tillhandahållna datasetet med webbtrafik och skapade ett nytt dataset som vi använde för att utvärdera riktigheten av tre WF attacker.Vid slutet hade vi samlat in 1345 filer med VPN-trafik. All trafik samlades in från den populära livestream plattformen twitch.tv. Livestreamingkanaler plockades ut från twitchs förstasida och vi slutade med 245 unika kanaler i vårat dataset. Med hjälp av vårat dataset lyckades vi minska noggrannheten för alla tre testade WF-attacker från 90% ner till 47% med tröskeln på 0,0 och från 74% ner till 17% med en tröskel på 0,99. Även om detta är en betydande minskning av noggrannheten kommer det med en ungefär tiofaldig ökning av antalet paket. I slutändan samlade vi bara trafik från twitch.tv men fick ändå några intressanta resultat och skulle gärna se fortsatt forskning inom detta område.Kod, instruktioner, dataset och andra artefakter finns tillgängliga via github.com/CSand/rds-collect.

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.

Mitochondriopathien sind Multisystemerkrankungen die durch verschiedene Defekte in den Energie (ATP) produzierenden Stoffwechselwegen der Mitochondrien verursacht sind. Will man Mitochondriopathien auf molekularer Ebene diagnostizieren, stößt man auf folgende Schwierigkeiten: (A) Ungefähr 1000 Gene sind an der Biogenese des Mitochondriums beteiligt. Die Dysfunktion jedes einzelnen dieser Gene kann potentiell zur Mitochondriopathie führen. (B) Mitochondriale Proteine werden durch zwei Genome, durch die mitochondriale und durch die nukleäre DNA kodiert. (C) Die klinischen Symptome der Patienten weisen selten auf die molekulare Diagnose, da der Phänotyp oft nur auf einem sekundären Energiemangel beruht. In der Regel besteht keine sichere Genotyp-Phänotyp-Relation. Mit den gegenwärtig zur Verfügung stehenden Methoden lassen sich bei nur 20% der Patienten Mutationen finden. Wir wollten daher eine neue Screening-Methode entwickeln, mit deren Hilfe wir hoffen, die Aufspürungsrate für mitochondriale Mutationen zu erhöhen. Die Gesamtheit der Proteine einer Organelle oder einer ganzen Zelle (ihr "Proteom") stellt das Verbindungsglied zwischen Geno- und Phänotyp dar. Aus diesem Grunde wollten wir das mitochondriale Proteom von gesunden Kontrollpersonen und von Patienten mit Mitochondriopathien untersuchen. Protein-Muster, die zwischen diesen beiden Gruppen abweichen, könnten die Aufmerksamkeit auf Gene und Proteine richten, die an der Entstehung des Krankheits-Phänotyps beteiligt sind. Um solch eine vergleichende Studie durchzuführen, muß zunächst eine Referenzkarte des normalen mitochondrialen Proteoms erstellt werden. In meinem Dissertationsprojekt habe ich diese grundlegende Arbeit durchgeführt und zahlreiche Proteine auf der Proteomkarte menschlicher Mitochondrien identifiziert, die aus Epstein-Barr-Virus-transformierten lymphoblastoiden Zellen gewonnen worden waren. Ich wählte diese Zellsorte als Untersuchungsmaterial, da sie nicht nur einfach von Patienten gewonnen werden, sondern auch potentiell permanent wachsen kann. Dies erlaubt die Züchtung einer hohen Zellzahl ohne übermäßigen Aufwand. Ich optimierte ein Protokoll zur Zentrifugation in einem hybriden Gradienten, mit dem genug gereinigte Mitochondrien aus 108 Zellen gewonnen werden konnten. Für die Referenzkarte benutzte ich die lymphoblastoide Zellline einer gesunden Kontrollperson. Die Methode der Wahl zur Proteinidentifikation in Proteom-Projekten ist die zweidimensionale Proteinelektrophorese gekoppelt mit der MALDI-TOF-Massenspektrometrie. Ich entdeckte mehr als 400 Punkte in meinem silbergefärbten zweidimensionalen Gel und analysierte die 141 stärksten Punkte nach in-gel Trypsin-Verdau und anschließender MALDI-TOF-Massenspektrometrie in einem Verfahren, das als "Peptide Mass Fingerprinting" (Peptidmassen-Fingerabdruck) bezeichnet wird. Mit Hilfe entsprechender Datenbanken konnte ich schließlich 115 verschiedene Proteinpunkte (entsprechen 95 verschiedenen Proteinen) identifizieren. 90 dieser Punkte (entsprechend 74 verschiedenen Proteinen) waren sicher mitochondrialer Herkunft und sind Komponenten aller wesentlichen im Mitochondrium lokalisierten Stoffwechselwege. 16 der 74 identifizierten mitochondrialen Proteine gehören zur Atmungskette. Obwohl 18 mitochondriale Proteine in der Datenbank SWISS-PROT als "Membran-assoziiert" annotiert sind, identifizierte ich nur vier Proteine mit sicheren Transmembrandomänen. Ich entdeckte keine der 13 durch die mitochondriale DNA kodierten Proteine, die alle stark hydrophobe Membranproteine sind. Andere Forscher sind bei dem Versuch diese Proteine zu identifizieren, auf die gleichen Schwierigkeiten gestoßen. Mit meiner Dissertationsarbeit habe ich unsere eigene Datenbank und Referenzkarte des mitochondrialen Proteoms lyphoblastoider Zellen erstellt. Diese Daten ermöglichen nun die Analyse des mitochondrialen Proteoms von Patienten. Meine weiteren Untersuchungen auf diesem Gebiet werden sich nun auf die genetische Variabilität des Proteoms gesunder Kontrollpersonen und auf das Proteom der Patienten mit Mitochondriopathien beziehen. / Mitochondrial disorders are multisystem diseases that can be caused by any defect in the energy (ATP) generating pathways in the mitochondria. The difficulty in diagnosing mitochondrial diseases on the molecular level arises from several obstacles: (A) About 1000 genes are involved in the biogenesis of mitochondria. The dysfunction of each of them may potentially cause mitochondriopathy. (B) The mitochondrial proteins are encoded by two genomes: the mitochondrial DNA and the nuclear DNA. (C) The clinical symptoms of the patients rarely suggest a molecular diagnosis since in most cases, the phenotype is a secondary phenomenon to energy depletion. Generally there is no genotype-phenotype relation. Based on current diagnostic methods in only 20% of the patients a mutation can be found. We therefore wanted to develop a new screening method by which we hope to increase the identification rate. Since the numerous proteins of an organelle or of a whole cell (its "proteome") connect the genotype with the phenotype, we set out to study the proteome of the mitochondrion in healthy individuals and in patients with mitochondrial diseases. Deviating protein patterns between the two individuals could direct the attention to disease-specific proteins and genes, which might be involved in the expression of a disease-phenotype. In order to perform such a comparison I first had to establish a normal reference map. In my dissertation project I performed this basic task and identified numerous mitochondrial proteins on the proteome-map of human mitochondria, which had been extracted from lymphoblastoid cells. I selected Epstein-Barr-Virus-transformed lymphoblastoid cells as samples not only because they are easily obtained from patients, but also due to their potential permanent growth. This approach allows the cultivation of high cell numbers without excessive expenditure of work and cost. I optimized a protocol for hybrid gradient centrifugation, by which enough mitochondria can be purified from 108 cells. I used a cultured lymphoblastoid cell line from a normal control patient and isolated mitochondria from it by using hybrid gradient centrifugation. In proteomics the combination of the high-resolution two-dimensional electrophoresis and matrix assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is currently the method of choice for protein identification. I detected more than 400 spots in a silver-stained two-dimensional gel. I analyzed the 141 strongest spots of it by trypsin in gel digestion and subsequent MALDI-TOF mass spectrometry in a process termed "peptide mass fingerprinting". After database search, I finally identified 115 protein spots (corresponding to 95 different proteins), 90 of which (corresponding to 74 different proteins) are of confirmed mitochondrial origin. These identified proteins are components of the main biological pathways located in the mitochondrion. 16 of the 74 identified mitochondrial proteins belong to the respiratory chain. Despite the fact that 18 mitochondrial proteins are annotated in the SWISS-PROT-database as "membrane associated proteins", only four of them have clear transmembrane domains. None of the proteins encoded by the mitochondrial DNA could be detected. All of them are hydrophobic membrane proteins. A similar difficulty in resolving these proteins was encountered by other research groups. With my dissertation I established our own database and reference map of the mitochondrial proteome of lymphoblastoid cells. These data will facilitate the analysis of the mitochondrial proteome in patients. My future research based on this dissertation will mainly focus on the genetic variation of the proteome of healthy individuals and on patients with mitochondrial diseases.

The State as the representative of the victims of crime is expected to protect those vulnarable group of people with due regard to the rights of the perpetrators’s of crime. It is imperative that the law of general application which is aimed at protecting victims of crime, be sufficiently effective to protect the victims. The Criminal Procedure Act 51 of 1977 is aimed at assisting the police to conduct pre-trial criminal procedure in order to bring perpetrators of crime to book. Sections 36A, 36B, 36C and 37 (both previous and as amended) of the Criminal Procedure Act including chapter 5A of the South African Police Act, 1995 are explored in this dissertation. This dissertation examines the areas in the Criminal Procedure Act that make it problematic for the police to conduct efficient and effective crime detection through the ascertainment of bodily features of the suspected or accused person. The law in three foreign jurisdictions relating to this topic are investigated and compared in order to make recommendations and suggest possible solutions. / Criminal & Procedural Law / LL.M.

Des études ont récemment mis en évidence le rôle des lipopolysaccharides (LPS) des bactéries à Gram négatif de la flore intestinale dans le processus de l’inflammation conduisant à l’obésité et au diabète de type 2.Le présent travail est réalisé dans le cadre d’une collaboration entre les équipes du Dr. Caroff (U. Paris-Sud, Orsay) et du Pr. Zhao (U. Jiao Tong, Shanghai). Les expériences présentées ont été réalisées lors de séjours dans les deux laboratoires.Il a été démontré en Chine que des bactéries Sulfato-réductrices (SRB) à Gram négatif étaient présentes en plus forte proportion dans la flore intestinale chez les souris suivant un régime gras. Les mêmes résultats ont été observés chez l'homme. L’hypothèse selon laquelle des SRB seraient à l’origine de grandes quantités d’endotoxines chez les obèses et les patients diabétiques a été émise. Plusieurs souches de SRB isolées de la flore intestinale humaine d’un sujet sain et d’un sujet diabétique ont été cultivées en Chine. Des études de relation structure/activité des LPS isolés de ces bactéries ont été réalisées dans le laboratoire Français pour déterminer leur rôle dans le développement des maladies métaboliques. Les souches isolées des deux sujets ont pu être classées dans le genre Desulfovibrio. Les LPS correspondants ont été extraits et purifiés par des méthodes mises au point dans l’équipe d’Orsay. La structure chimique a été élucidée par les méthodes suivantes : Electrophorèse, Chromatographie sur couche mince, Chromatographie en phase gazeuse et Spectrométrie de masse MALDI. C’est ainsi que des spectres de masse ont été obtenus et que la structure des lipides A, principes actifs des LPS, isolés de SRB a été décrite pour la première fois. Les activités biologiques testées (TNFα, IL-6) varient en fonction du nombre d’acides gras présents. Les LPS de SRB du patient sain ont une structure variable (Smooth versus Rough) en fonction de la quantité de fer présent dans le milieu, et ceux isolés du patient diabétique présentent des structures atypiques qui ne sont pas toutes inflamogènes. Une molécule membranaire inconnue, que nous avons nommée « Glycosyl’X » était co-extraite avec les LPS. Elle joue apparemment un rôle important dans la croissance des SRB et a été étudiée après des étapes de purification complexes. Les structures et le pouvoir inflammatoire de ces molécules dont la structure varie avec les souches, et qui chélatent le fer, ont été étudiées. Elles sont de nature principalement osidique et fixées à la membrane. La proportion de ces molécules par rapport aux LPS varie avec la quantité de fer disponible dans le milieu. Un milieu riche en fer favorise la croissance des Desulfovibrio portant les Glycosyl’X qui n’ont pas de pouvoir inflammatoire eux-mêmes, mais entrent en compétition avec les LPS, modulant ainsi indirectement l’activité de ces derniers. L’augmentation du nombre de Desulfovibrio conduisant à l’augmentation des molécules Glycosyl’X pourrait aussi moduler positivement (par présentation) ou négativement (par élimination des bactéries) l’adsorption du fer dans les intestins dont l’équilibre est essentiel pour l’homéostasie métabolique.Par ailleurs, la croissance des Desulfovibrio augmente la production d’Hydrogène Sulfuré connu pour son action délétère sur les cellules. Nous favorisons l’hypothèse selon laquelle son action sur la disjonction des cellules épithéliales permettrait le passage des différents LPS relargués par la flore Gram-négative intestinale, et même des bactéries entières, vers la circulation sanguine. / Recent studies have highlighted the role of lipopolysaccharide (LPS) in the intestinal flora (gut microbiota) which could contribute to the inflammation process leading to obesity and type 2 diabetes. This thesis is part of a collaborative project between the laboratories of Dr. Caroff (U. Paris -Sud, Orsay, France) and Prof. Zhao (U. Jiao Tong , Shanghai, China). It has been shown by Pr.Zhao’s team in 2010 that the Sulfate -Reducing Bacteria (SRB) were presented in greater proportion in the intestinal mice flora following a fat diet compared to mice following a normal diet. The same results were observed in humans. The starting hypothesis was that SRB could produce a large amount of endotoxin in obese and diabetic patients and play a role in the development of metabolic diseases. Several SRB strains isolated from the human intestinal flora of a healthy subject and of a diabetic subject were grown in the Chinese laboratory. Studies of their LPS structure / activity relationships were carried out in the French laboratory. The aim of this study was to determine their roles in the development of metabolic diseases.Strains isolated from the two subjects could be classified in the Desulfovibrio genus. The corresponding LPS were extracted and purified by the methods developed in the French laboratory. The chemical structure was elucidated by the following methods: Electrophoresis, Thin layer chromatography, Gas chromatography and MALDI mass spectrometry. The mass spectra were obtained and the structure of lipid A, the active part of LPS isolated from SRB was described here for the first time. The biological activities test (TNFα, IL-6) vary depending on the number of fatty acids present in their lipid A structure. The LPS of SRB isolated from the healthy patient had a variable structure (Smooth versus Rough) depending on the amount of iron present in the medium, and those isolated from diabetic patients had atypical structures are not all inflamogenic .An unknown membrane molecule, which we named "Glycosyl'X" was co-extracted with the LPS. It apparently plays an important role in the growth of SRB was investigated after complex purification steps. The structures and the inflammatory power of these molecules variying with strains chelating iron were studied. They are mainly of glycosidic nature and linked to the bacterial membrane.The proportion of these molecules relatively to LPS varies with the amount of iron in the medium. An environment rich in iron promotes the growth of Desulfovibrio Glycosyl'X, molecules but competes with LPS and indirectly modulates the activity of the latter. The increase number of Desulfovibrio leading to increased Glycosyl'X molecules may also modulate positively (by presentation) or negatively (by killing bacteria) the absorption of iron in the intestines which balance is essential for metabolic homeostasis.Furthermore, the growth of Desulfovibrio increasing the production of Hydrogen Sulfide is known for its deleterious effects on the cells. We favor the hypothesis that its action on the separation of epithelial cells favors the passage of different LPS released by the Gram- negative of intestinal flora and even whole cell bacteria into the bloodstream.