A pilot-scale, two-stage (carbon oxidation stage one, ammonia oxidation stage two) fixed film biological aerated filter (BAF) process was operated on-site at a domestic wastewater treatment plant. Over the study period, hydraulic loadings to the system were varied, generating a range of organic and ammonia loading conditions. Nitrification was monitored along the length of the filters by measuring chemical nitrogen species and activity levels of ammonia oxidizing bacteria (AOB). During the first phase of the study, nitrification performance was characterized during the wintertime and compared with oligonucleotide probing results using an ammonia-oxidizer specific probe. Overall nitrification efficiency for wintertime conditions (average temperature 12.4 ± 0.1°C) was greater than 90 percent when ammonia-N loadings to the second stage were 0.6 kg/m3-day or less. Nitrification efficiency started to deteriorate at loadings beyond this point. Biofilm and liquid samples were collected along the distance of the two columns at high and low ammonia loadings. The degree of activity observed by ammonia oxidizing bacteria in the biofilm corresponded with the disappearance of ammonia and the generation of nitrate as water passed through the columns. During the second phase of the study, the probing methods were investigated and results of two approaches of analysis were compared to chemical nitrogen profiles. It was found that probe signals normalized to mass of total bacterial nucleic acid corresponded better with chemical profiles than using a novel method of standardizing against known nucleic acid mass standards. During both phases of the study, zones of ammonia oxidizing activity progressed along the length of the columns as organic and ammonia loadings to the system increased. The oligonucleotide probe data suggest that this shift in the location of the nitrifier population is due to higher BOD loads to the second stage, which supported higher levels of heterotrophic growth in the second stage of the system. It was concluded that the strongest influence on nitrification performance in this type of BAF system is likely to be competition between heterotrophs and autotrophs. / Master of Science

Le cancer de la prostate (CaP) est actuellement le cancer le plus fréquent en France et constitue l'une des principales causes de décès par cancer chez l'homme dans les pays industrialisés. Un tiers des patients avec un CaP à priori localisé auront déjà des micro-métastases au moment du traitement local. Ces patients qui répondent dans un premier temps à la castration (hormonothérapie) seront cependant en échappement hormonal dans les 2 ans qui suivent. Récemment, plusieurs essais cliniques de phase III ont rapporté un gain de survie avec la chimiothérapie à base de docétaxel dans les CaPs métastatiques résistants à la castration. Néanmoins, la survie n'est prolongée que de 2 ou 3 mois et de nouvelles approches thérapeutiques ciblant des voies de signalisation spécifiques sont donc nécessaires. Les travaux réalisés au cours de cette Thèse ont permis tout d'abord de montrer, grâce à l'utilisation de TMAs, que la surexpression de TP53INP1, une protéine de réponse au stress, était un facteur de mauvais pronostic dans le CaP, prédictif notamment du risque de rechute biologique. Nous avons ensuite pu montrer grâce à des xénogreffes de cellules tumorales (LNCaP) que les taux d'ARNm de TP53INP1 diminuaient durant l'hormonothérapie et que TP53INP1 était de nouveau significativement surexprimée dans les tumeurs résistantes à la castration. Nous avons développé et déposé un brevet pour un oligonucléotide antisens (ASO) inhibant TP53INP1. Le traitement in vitro des lignées cellulaires hormonosensibles LNcaP et hormono-résistantes C4-2 par l'ASO induit une diminution d'expression de la protéine TP53INP1, inhibe la prolifération cellulaire et induit une augmentation de l'apoptose. / Prostate cancer (PC) is the most common malignancy in France and one of the most frequent leading causes of cancer-related death in men in industrialized countries. Even with aggressive screening, approximately one-third of patients believed to have localized PC will already have micro-metastatic disease at the time of definitive local therapy. These patients initially respond to androgen ablative therapy, but with time, their tumors ultimately become unresponsive and recur within 2 years as castration-resistant prostate cancer (CRPC). Recently, docetaxel-based regimens have shown improved survival in men with CRPC in phase III studies. However, the median overall survival was prolonged for only 2-3 months, and thus development of new therapeutic approaches that target relevant signaling pathways are essential to restore the androgen-sensitivity of CRPC. We showed, using tissue micro-array (TMA) analysis, that over-expression of Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1), a cell stress response protein, is a worse prognostic factor in PC, particularly predictive of biological cancer relapse. We also we found that TP53INP1 protein expression decreases during castration therapy (CT) and significantly increases in human CRPC. TP53INP1 mRNA was also significantly increased in castration-resistant (CR) tumors of LNCaP xenograft compared to the castration-sensitive (CS) taken before CT. We developed and world-wide patented one antisense oligonucleotide (ASO) targeting TP53INP1 (PCT/IB2011/054555). Treatment of LNCaP and C4-2 cells in vitro with TP53INP1 ASO downregulates TP53INP1 protein level, inhibits proliferation and induces apoptosis.

Polynuclear platinum compounds represent a new class of potential platinum anticancer therapeutics. Derived from the most widely used platinum anticancer drug, cisplatin, these novel compounds are distinct in their interactions with bio-molecules. The effectiveness of platinum anticancer agents is influenced by three pharmacological factors: (i) their resistance to deactivating sulfur nucleophiles, (ii) the ability to gain cellular entry and efficient cellular uptake, and (iii) the ability to form stable and specific complexes with DNA. BBR 3464, the first multinuclear platinum compound to reach phase II clinical trials, has created a new approach to cancer drug design. Large, highly charged platinum compounds have been shown to form favorable covalent and noncovalent interactions with bio-molecular structures. Compounds such as BBR 3464, form an immediate pre-association with anionic structures on biomolecules before covalent attachment. To better characterize these interactions, a new set of compounds was designed that exclusively interacts via electrostatic associations and hydrogen bonding. The investigation of noncovalent complexes between DNA, proteins, and peptides with a variety of synthetic and biological relevant structures has become increasingly more common with the coupling of electrospray ionization and mass spectrometry (ESI-MS). Mass spectrometry has been useful to the drug design community by allowing the rapid and accurate characterization of drug binding sites. In the first project we have explored the use of collision induced dissociation (CID) to map the potential binding sites of noncovalent polynuclear platinum compounds of varying size and charge with an antisense oligonucleotide of the Bcl-2 sequence. In the second project, the gas-phase dissociation and stabilizing effects of these polynuclear platinum compounds on duplex DNA were determined. Correlations between the size and charge of associating platinum compounds were determined by comparing the change in gas phase stability under CID conditions. Additionally, the association of these new types of noncovalently binding polynuclear platinum compounds was investigated with model cell surface structures such as anionic heparan sulfate and phospholipids.

Die vorliegende Dissertation leistet einen Beitrag zur spektroskopischen Messmethodik nanoskaliger Strukturen. Im Mittelpunkt der Arbeit steht die Entwicklung und Erprobung eines spektrofluorimetrischen Aufbaus, mit dessen Hilfe ein aus Kohlenstoffnanoröhren und DNA-Oligomeren bestehendes supramolekulares Modellsystem einer optischen Untersuchung zugänglich gemacht wird. Die Vielseitigkeit der Messeinheit aus Mikroskop und Spektrometer wird an einer weiteren Substanzklasse untermauert. So wird das Emissionsverhalten von in Siliziumcarbidkristallen induzierten Defektzentren einer räumlich, spektral und zeitlich aufgelösten Charakterisierung unterzogen. Die zentrale Komponente des Spektrofluorimetrieaufbaus stellt eine Superkontinuumlichtquelle dar. In Verbindung mit einem elektronisch geregelten Filtermodul zur Wellenlängenselektion erlaubt sie die Durchführung von Photolumineszenz-Anregungsexperimenten. Im Gegensatz zu kommerziell erhältlichen Systemen, die überwiegend auf eine spektroskopische Charakterisierung gelöster oder kolloidal stabilisierter Substanzen abzielen, erlaubt der hier realisierte Aufbau auch die PL- mikroskopische Untersuchung kondensierter Proben, was durch die Epi-Bauweise auch opake Substrate einschließt. Der Einsatz von InGaAs-Sensoren weitet das Detektionsfenster auf den Nahinfrarotbereich aus, sowohl hinsichtlich des Kamera- als auch des Spektroskopiekanals. Anhand verschiedenartiger Kohlenstoffnanorohrproben, die entweder in flüssiger Phase dispergiert oder in festem Zustand als Film abgeschieden vorliegen, wird die Leistungsfähigkeit des PLE-Experiments unter Beweis gestellt. Neben der Zuordnung der Chiralitäten in polydispersen SWNT-Suspensionen wird dies auch durch die Untersuchung von Energietransferprozessen und die Studie von Umgebungseinflüssen demonstriert. Die Charakterisierung des DNA-SWNT-Modellsystems in mikrofluidischer Umgebung macht von der fluoreszenzmikroskopischen Detektionseinheit Gebrauch. Während die intrinsische Photolumineszenz der Nanoröhren sicherstellen soll, dass Letztere in ausreichender Anzahl auf den mikrostrukturierten Substraten vorhanden sind, wird die extrinsische Photolumineszenz der funktionalisierten Oligonukleotide als spektroskopisches Maß für die DNA-Konzentration herangezogen. Das hierbei beobachtete Agglomerationsverhalten der farbstoffmarkierten Oligomere geht mit einer lokal erhöhten Fluoreszenzintensität einher und erlaubt damit die quantitative Auswertung der auf PL-Einzelbildern basierenden Zeitserien. Zugleich wird damit eine Abschätzung der DNA-Belegung auf den Nanoröhren möglich. Im Falle der aus 16 alternierenden Guanin-Thymin-Einheiten bestehenden Basensequenz lösen sich nach Initiieren des Desorptionsvorgangs ein Großteil der Oligomere von der Nanorohroberfläche ab. Lediglich ein Fünftel bleibt in adsorbierter Form zurück, was sich jedoch für die Hybridstabilität als ausreichend erweist. Die Freisetzung weiterer Oligomere bleibt bei der Versuchstemperatur von 20 °C trotz der hohen Verdünnung aus, da aufgrund des größeren Interadsorbatabstands und der damit verbundenen Abnahme repulsiver Wechselwirkungen die Aktivierungsbarriere für ihre Desorption steigt. Die Stabilität der DNA-SWNT-Konjugate liegt demnach in ihrer kinetischen Inertheit begründet, die sie vor einer Reaggregation bewahrt. Die Studie der in Siliziumcarbid induzierten Fehlstellendefekte kann als Beleg für die breite Anwendbarkeit des spektrofluorimetrischen Aufbaus gelten. PL-Mikroskopaufnahmen zeigen hierbei, dass die Anzahl der Defektzentren mit der Bestrahlungsintensität kontrolliert werden kann – von einer kontinuierlichen Verteilung bei hohen Strahlungsintensitäten über heterogene Defektansammlungen bis hin zu Einzeldefektstellen bei niedrigen Strahlungsdosen. Letztere resultieren in beugungsbegrenzten Signaturen und erlauben damit eine Charakterisierung des abbildenden Systems sowie des Anregungsfokus. Anhand der PLE-Analyse lässt sich das Absorptionsmaximum abschätzen. Aussagen zur zeitlichen Entwicklung des Emissionsverhaltens werden durch TCSPC-Messungen erhalten. Die abschließende Untersuchung des Photonenflusses mit Hilfe von Korrelationsexperimenten nach Hanbury Brown-Twiss zeigt bei Raumtemperatur kein Auftreten von Photonantibunching. / Within the scope of this dissertation, a contribution towards the spectroscopic investigation of nanomaterials has been made. The approach applied here is a spectrofluorometric one, which allows the optical characterization of an oligonucleotide/single-wall carbon nanotube comprised supramolecular model system. The flexibility of the developed setup is demonstrated by studying another class of nanoscale samples, that is defect centers in silicon carbide crystals. Their emission behavior is subject to a spacial, spectral and temporal analysis. The key role in the combined microscope and spectrograph assembly is held by a supercontinuum light source. With the help of this device, excitation measurements can be conducted by shifting the wavelength with an electronically driven filter accessory. In contrast to commercially available systems, which predominantly focus on a spectroscopic characterization of substances in solution or in colloidal suspension, it is also possible to carry out PL microscopic studies of condensed matter. Because of an epifluorescence configuration, the samples to be measured imply opaque substrates as well. Using complementary sensor materials including InGaAs arrays enlarges the accessible range of emission for both imaging and spectroscopy. Differently processed carbon nanotube samples, occurring in either dispersed or deposited form, serve as a benchmark in assessing the capability of the PLE setup established here. For instance, it can be used to assign chiralities in heterogeneous SWNT suspensions or to analyze energy transfer as well as the impact of varying colloidal conditions. The studies of the DNA-SWNT model system are accomplished through the use of fluorescence microscopy under microfluidic control. The intrinsic photoluminescence of carbon nanotubes can be exploited to estimate, to what extent they cover the lithographically treated silicon wafers. The extrinsic photoluminescence of functionalized oligonucleotides is used as a spectroscopic probe for DNA concentration measurements. Bright spots with distinct shape are observed and attributed to an agglomeration of dye-labeled oligomers. By recording time series of PL images, the locally enhanced emission signal in these discrete sites can be quantitatively analyzed, representing the progress of DNA adsorption on SWNTs. Based on a DNA sequence consisting of 16 alternating guanine-thymine moieties, the present experiments reveal the release of most of the oligonucleotides, when starting off the desorption process. Only one fifth of the initially adsorbed amount remains attached to the nanotube surface, without the modified environment affecting hybrid stability. Remarkably, an ongoing desorption does not take place at the test temperature of 20 °C in spite of the vast dilution applied. This circumstance can be explained by an increased distance between the residually adsorbed oligonucleotides, resulting in less pronounced repulsive forces between them. Consequently, the activation energy barrier for inducing further detachment is raised. In case of sufficiently long base compositions, this suggests that the stability of conjugates is founded in their kinetic inertness. The absence of continued desorption eventually prevents these DNA-SWNT hybrids from reaggregating. The investigation of vacancy defects in silicon carbide proves the broad applicability of the spectrofluorimetric setup. PL microscopic studies show that the amount of defect sites can be controlled by tuning electron irradiance. The corresponding defect pattern evolves from a continuous distribution towards discrete clusters. By lowering the exposure dose even more, single defects emerge showing diffraction-limited signatures, which can help to elucidate the imaging system as well as the excitation focus in more detail. PLE mapping and time-correlated single photon counting of the fluorescence decay provide insight in photophysical parameters, including the absorption maxium and the lifetime of the excited state. Studying the photon flux by means of correlation measurements according to Hanbury Brown-Twiss does not give rise to photon antibunching under ambient conditions.

The plasma membrane functions as a barrier, restricting entry of hydrophilic pharmaceutical agents. Cell-penetrating peptides (CPPs) are capable of transporting bioactive cargos into the cell and have consequently been extensively investigated for their mechanism of entry and capability to deliver various cargos spanning from peptides to plasmids. The main aim of this thesis was to investigate the mechanism and capability of some of these CPPs to deliver mainly oligonucleotides and peptides into the cell. Oligonucleotides in the form of ds DNA decoy for sequestering of transcription factors or PNAs for redirection of splicing. In addition, peptides derived from the interaction interface of a tumor suppressor protein were investigated for their potential to combine a biological effect with internalization. Peptides with or without any cargo were predominantly dependent on some form of endocytic mechanism for internalization, substantiated by using a functional assay, where all tested CPPs were associated with endocytosis for delivery of splice correcting PNAs. A new CPP, M918 proved most efficient in promoting splice correction and internalized mainly via macropinocytosis. In addition, TP10 efficiently delivered dsDNA decoy oligonucleotides for sequestering of the transcription factor Myc with a concomitant biological response, i.e. reduced proliferation. Finally, for the first time, to our knowledge, a novel pro-apoptotic peptide with cell-penetrating properties was designed from the tumor suppressor p14ARF, which decreased proliferation and induced apoptosis in cancer cell-lines, potentially mimicking the full-length protein. Altogether, this thesis highlights the functionality of CPPs and the possibility to develop new CPPs with improved or new properties, having the potential to advance delivery of therapeutic compounds.

High molecular weight biomolecules are becoming important in the development of new therapeutics. However, their size and nature creates a major limitation for their application – poor penetration through biological membranes. A new class of peptides, cell-penetrating peptides (CPPs), has shown the capability to transport various macromolecules inside the cells. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity and restricted bioavailability resulting from endocytic uptake of CPPs and entrapment in endosomal compartments. This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to achieve in vivo selectivity towards cancer cells. The first two papers employ tumor homing peptides as targeting moieties coupled to the N-termini of CPPs. In the third paper, a CPP is C-terminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific cleavage site followed by an inactivating amino acid sequence. In tissues overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated after proteolytic removal of the inactivating sequence, thus the cargo can be transported inside the cells. In paper IV, several CPPs have been N-terminally modified with a stearyl moiety and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide delivery as Lipofectamine™ 2000. Moreover, stearyl-TP10 has preserved efficacy in serum and is not toxic to cells. In conclusion, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.

The study of the mechanism of action of an antitumor active drug is essential for improving the efficacy and reducing the side effects of the drug as well as for developing better alternatives. In this vein, reactions of dirhodium compounds with DNA oligonucleotides were investigated by the techniques of mass spectrometry, HPLC, and NMR spectroscopic analytical methods. The relative reactivities of three dirhodium compounds, namely Rh2(O2CCH3)4, Rh2(O2CCF3)4, and [Rh2(O2CCH3)2(CH3CN)6](BF4)2, with DNA oligonucleotides were studied and compared to the clinically used anticancer drugs cisplatin and carboplatin using both MALDI and ESI mass spectrometric methods. The compound Rh2(O2CCF3)4 exhibits the highest reactivity among the dirhodium compounds, which is comparable to cisplatin, followed by [Rh2(O2CCH3)2(CH3CN)6](BF4)2, and finally Rh2(O2CCH3)4 which is the least reactive. Various dirhodium-oligonucleotide adducts were detected with both MALDI and ESI methods, which involve substitution of different numbers of the original ligands of the given dirhodium compound. ESI MS was found to be a sufficiently soft ionization method for detecting intact metal adducts, and CID MS-MS was useful for detecting weakly bound species such as axial adducts [M+Rh2(O2CCH3)4] and for comparing the relative bond strength between ligands in the metal adduct. A combination of anion exchange HPLC purification and enzymatic digestion studies of the adducts of Rh2(O2CCH3)4 with the 5'-CCTTCAACTCTC oligonucleotide revealed that Rh2(O2CCH3)4 binds to the center or to the ends of the oligonucleotide sequence by displacement of one or two acetate groups. Kinetic products of the type [M+Rh2(O2CCH3)3] obtained from the reaction of Rh2(O2CCH3)4 with 5'-CTCTCAACTTCC were separated by employing both reverse phase and anion exchange HPLC methods. The adduct that involves binding of the dirhodium unit to the exocyclic N4 atom of C5 and the N7 of A6 was found to be most stable whereas other adducts involving binding of C3 or C12 residues are clearly less stable. Reaction of cis-[Rh2(DAP)(O2CCH3)3(CH3OH)](O2CCH3) (DAP = 1,12- diazaperylene) with 5'-CTCTCAACTTCC produced a major adduct in which DAP group intercalates between 6A and 7A in the double stranded adduct with the rhodium atom that is not coordinated to the DAP group forming a covalent bond to the N7 atom of 6A which lends stability to the adduct.

高密度寡核甘酸基因陣列實驗是新的生物技術，可在一個晶片上蒐集到數千至上萬個基因資料，資料處理的過程相當繁複，包括背景訊號的修正、正規化、探針背景的修正及探針組資料的整合，本研究首先將介紹各資料處理步驟。其中正規化的目的是要修正資料中由實驗產生的系統化變異，去除實驗誤差，使資料更為純淨，則後續所做的統計分析才會更為精確。之後再詳細介紹三種正規化方法，包括：尺度調整法、循環平滑調整法及百分位調整法。並將以一組實際資料來說明正規化後的結果。最終採取電腦模擬的方式，以平均四分位距、平均標準差、Diff統計量及離群值的個數這四個量化準則，來研究各正規化方法的效果，以及比較這三種正規化方法的優劣，同時也將探討此四種準則的適當性。 / High-density oligonucleotide array gene experiment is a new biological technology. More than thousands of gene data can be obtained in an array. The data processing includes background correction, normalization, probe specific background correction and summarizing the probe set value into one expression measure. The goal of normalization is to remove the systematic variation induced in the experiment while keeping the biological variation of interest. Using the purified data, one will obtain more accurate conclusions in subsequent statistical analysis. Firstly, we introduce the data processing procedures. Three normalization methods, which include Scaling, Cyclic Loess and Quantile, are explained in detail and illustrated by a real data set. Moreover, a simulation study is conducted to compare the three methods. Four quantities, Mean of IQR, Mean of Standard Deviation, Diff Statistics and Outlier, are proposed for assessment. Not only the performances of the three normalization methods but also the properties of the four proposed criteria are given and studied in this research.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Diss. Stockholm : Karol. inst., 2004.