want this O Super Silva Ebook. You should get the file at onceHere is the working pdf download link for O Super Silva Ebook. Image not found or type unknown. The Super-Wicked Problem of Antimicrobial Resistance Jasper Littmann, PhD U.K. [email protected] Diego S. Silva, PhD Assistant Professor Faculty of .. 1 J O'Neill, et al., Infection Prevention, Control and Surveillance: Limiting the. “I own a little bar and I've got to be there when the next shift comes on at two o' clock. Why don't you come along for the ride?” He didn't have to ask twice.

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Not your computer? Use Guest mode to sign in privately. Learn more. Next. Create account. Afrikaans . azərbaycan . català . Čeština . Dansk . Deutsch . eesti. Dissertação de Mestrado apresentada por Robson dos Santos e Silva à Pós- Graduação em Ciência da Pernambuco, sob o título “A rigorous methodology for developing GUI-based DSL name. In PL, the additional keyword from is necessary to state that a variable of a super OCL//PDF. acessed jan/ Bolt O Super usaascvb.info - Download as PDF File .pdf), Text File .txt) or read online. Bolt O Super usaascvb.info Uploaded by Edlene Luciana da Silva. Copyright: © All.

Structural biology Abstract The dynamic behaviour of supramolecular systems is an important dimension of their potential functions. Here, we report on the use of stochastic optical reconstruction microscopy to study the molecular exchange of peptide amphiphile nanofibres, supramolecular systems known to have important biomedical functions. Solutions of nanofibres labelled with different dyes Cy3 and Cy5 were mixed, and the distribution of dyes inserting into initially single-colour nanofibres was quantified using correlative image analysis. Our observations are consistent with an exchange mechanism involving monomers or small clusters of molecules inserting randomly into a fibre. The results reported here show that peptide amphiphile supramolecular systems can be dynamic and that their intermolecular interactions affect exchange patterns. This information can be used to generate useful aggregate morphologies for improved biomedical function. Introduction Reversible supramolecular interactions are ubiquitous in nature, controlling the self-assembly of ordered functional structures that need to be dynamic to perform their biological functions. One-dimensional cytoskeletal filaments such as actin and tubulin are typical examples of structures that use dynamics to mediate the adaptive behaviour of cells, resulting in cell motility, shape change, cell division, signalling and muscular contraction at larger length scales 1 , 2 , 3 , 4 , 5 , 6. Artificial supramolecular materials could offer this bio-inspired dynamic behaviour, thus allowing enhanced interaction with natural systems and increased biomedical functionality.

It was a sneer—no, more intense than a sneer. I give you every chance in the world and you tell me no. The first thing you do is get the money. Now strip off your clothes. I suddenly realized that Chuck was crazy, really insane, that he actually expected me to take off my clothes and go out there to have sex with five strangers.

When he took his hand out of his trouser pocket, he was holding his pistol and pointing it at me. You want to know what I think? You think they care what happens to some nickel-and-dime hooker who has an accident in some motel room? You think one of those guys would say he was out there waiting for a prostitute? And that about sums it up for you, too. Take off your clothes or you are one fucking dead chick!

I knew he would shoot me. I was numb as I removed my clothes and put them on hangers. Then tears started to flow out of my eyes. I was trembling, really shaking, too scared even to pray. When he realized he had triumphed, his attitude went from menacing to superior and condescending.

But this day, this hour, this moment—it was the turning point of my life and I have relived it again and again. When I look back, I always ask myself whether there had been any indications, any clues, any warnings. There were no hints that I was about to cross over from day to night.

I remember the details with awful clarity; I remember that room as if I had never left it, as if I were still there. And now, as always, I asked myself one question: Even today I think yes —yes, he would have killed me on the spot. And then I ask myself this: Would I go through it all again? No, today I would take the bullet. I go back to that moment and I plot out escape scenarios, things that might have been done and things I might have said.

I might have taken off my clothes, walked out into the room and then raced for the door. I might have picked up an ashtray and thrown it through the window and screamed for help. I might have hidden behind one of the men. Two of the men waiting for me were also naked. The other three were partially undressed. There was nothing in my imagination that could equal what was actually happening to me. I had a sudden vision of the barmaid at the Vegas Inn, the one with three men at the same time, and tears started to roll down my cheeks.

The man who had first greeted Chuck at the door now took him off to one side. I opened my eyes and I saw Chuck staring at me, his face full of hatred. The man who had led me over got on top of me and entered me with no preliminaries whatsoever. The second man who was naked came over and put his thing in my mouth, just like that, no words, no explanations. He was stiff and he guided himself into and out of my mouth.

By now, a third man had taken off his shorts and was coming over to the bed. He reached down, took my hand, and put it on his thing. You just go on sucking. I had the feeling that this was no more exciting for them than it was for me; they were robots with a robot. They would busy themselves for a while at one spot, then change positions.

One of the other men was still talking to Chuck, still complaining that my attitude was not all that it might have been. The fifth man—he was about thirty-five with blond hair parted on the side—was not taking part. He stood near the other bed and most of the remarks were sent in his direction. Jim remained quiet and his eyes seemed sad. For a while, I almost liked him, but then he joined the party, as did the fifth man. Chuck went over to the window beside the front door and remained there, looking out, standing guard.

My first thought was that we were going to stop and get something to eat. By this time I had lost all appetite. The man who had been calling for the sandwich lay on his back and the others put me on top of him. Then I felt another man climbing on my backside. I understood then that they were talking about a human sandwich. I had never experienced anal sex before and it really ripped me up.

I began to whimper. He was the top half of the sandwich. The three animals who had come on first cared about nothing but getting their jollies.

I can no longer remember their faces. They never talked to me directly. Most of the time my eyes were tightly closed. They picked me up and moved me here and there; they spread my legs this way and that; they shoved their things at me and into me.

Three of the animals were constant and persistent, always coming at me, not even resting between times. The other two would back off from time to time. Two of the men got their biggest thrill by working themselves up to the point of coming and then shooting their sperm all over my body and rubbing it in.

I had never been so frightened in my life. Every time I looked over at Chuck, his look scared me all over again. I was scared by what was happening to me at that moment and what might happen to me next. I had no idea what they were talking about. But two of the men tried to pry their way ino me at the same time.

A lot was still happening to my body but it stopped meaning anything to me. My breasts were being mauled and I stopped feeling that. It was as if my body belonged to someone else. Finally they began to tire and to take occasional breaks. Maybe they were getting bored.

After all, I had only so many hands and only so many openings and before too long, all possibilities were exhausted. Then Chuck came over to the bed and looked down at me. I had never wanted a shower so much, and I had never scrubbed myself so hard. I scrubbed at my skin where they had come all over me. Then I scrubbed at the rest of me. I wished I could melt into the shower drain and disappear.

I was filled with hurt. And I kept turning to God. As far as I was concerned, it was His fault. He had put me here.

All the time I was in the shower, I was talking to God. Please tell me why. He did help me get through it. He did help me survive. So I guess He was helping me out after all. But it took me a long time to come to that belief. When I went into the room to dress, the men were gone and Chuck was counting out the money on the bed.

The one who had been complaining about my attitude had demanded a refund and Chuck had given him back half his money. I was still speechless and in shock.

I had no idea that human beings did such things. I knew that a prostitute offered sex for money, but somehow I figured they would make love—that there would be kissing and caressing and some gentleness. I followed him out of the room and to the car. The minute we were in the car, with the doors closed, he turned to me and started yelling.

What the fuck is it with you anyway? You better start getting your shit together, Linda. I could still feel those hands all over me, pressing me, squeezing me, milking me. You know the difference between you and a pro? The way you were, they hadda do all the fucking work. I went over exactly what had happened. Good God! We can write them off. You know what I was trying to do today? I was trying to get a business started. Our business. Just trying to get a business started.

You know how you fucking get a business started? Then each of them goes out and tells two more people. Then you got ten, fifteen, guys. And they talk it up and then you got a little business going there.

You know what these guys are going to tell their friends after today? Next time better be different. Next time better be better. Oh, God, what next time? The following day, Chuck introduced me to a visitor, an old friend who managed a truck-rental business, and my name became Tracy at this point. I was alone with my first paying customer, my first trick. Harry thumbed through his wallet and fished out two twenty- dollar bills.

Just like old times. He was soft- spoken and that has always been an important quality with me. But at this moment he was not even on my mind that much; I was hardly thinking about him. I could think of only one thing— Chuck had finally left me alone. Well, not entirely alone. There was one matter to take care of first.

I took off my jeans and blouse and laid them over a chair.

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Was I supposed to take off the rest or was he? I had no idea. I went over to the bed and sat down on the edge of it and waited for him to come over and do whatever he was going to do to me.

But the thing is, you should at least be friendly. In fact, you should fake like you really want it. I was scared and I was dry but Harry went right into me.

But he went on anyway, pumping away. I lay there lifeless, letting him do all the work. Before too much longer, I would learn to fake it, even to be aggressive, just like the other hookers, but right then I just wanted him to finish and get off me. The moment he was done, I rolled out from under him and went to get my clothes.

But finally he stopped laughing, got up, got himself dressed and got himself out the front door. I counted to ten, then followed him.

As I opened the door, I heard a small noise behind me. A hand on my shoulder. He had been standing there in the hallway the whole time.

Then he started punching my body over and over again until I collapsed on the floor. No fucking good! Every day I either got raped, beaten, kicked, punched, smacked, choked, degraded, or yelled at.

Sometimes, I got all of the above. Strangely enough, what bothered me most was the endless verbal abuse. He never let up: I was so dumb; I was so ugly; I was so fat; I was so thin; I was so flat-chested, and I was so lucky to have him taking care of me.

The constant yelling took everything out of me. I had as much to do with the money as a teller at a bank; I got it from one man and passed it along to another man. That was the end of my contact with any money. These financial transactions would occur three times on an average day.

Before long, as his business grew, Chuck was able to add to his staff. The first arrival was a young girl named Moonshine. Moonshine was strictly a volunteer. She had been making love with a married man who had been paying her rent. A second boyfriend started taking care of her telephone bill. There was someone else to pay the electric bill and a fourth man who gave her a rented car to use.

Before long, Moonshine had many steady visitors and no bills to pay. She came to Chuck with the idea of expanding her horizons and perhaps even getting some take-home pay. At any rate, Moonshine was there to share the work load. Then came Debbie. And Melody. Now you might think that this would take some of the pressure off me. But there was more to it than that.

You see, Chuck had his own system of distributing the tricks. If a customer was handsome or clean-cut or just young, Chuck would send him off with one of the other working girls. He would fix me up with creeps and degenerates; he would watch them rape me through a hole in the mirror; but he would bristle with jealousy if a young or good-looking man paid any attention to me. You are jealous.

And, in truth, I began to forget what normal people with normal feelings were like. Lines like: And then there was Greg. Greg was thirty-eight years old, a successful architect, and he hired two of us. Greg had a slightly different script. What are you doing with this cheap girl?

I guess this is where I got my early dramatic training. All I had to do was take off my clothes and take a bubble bath in one of those circular sunken tubs. The trick sat and watched me soap myself for a full hour and that was the extent of our involvement. I had no idea how I was supposed to react to this sort of thing. Before he left, the trick complained to Chuck about my amateurishness. Also to a brand new line of verbal abuse: One trick who lived alone on a houseboat asked me to sit on his face and urinate in his mouth.

The trick got angrier and angrier, and, finally, he screamed at me to go into the bathroom and put it in a glass. I returned with a glassful of urine and he drank it down. Then he told me to get off his boat and never come back. I was happy to oblige.

And there was always the possibility of escape, the thought that he might slip up and leave me unguarded for a few minutes. Still and all, the trips from home were never what you might call pleasure trips. For one thing, I never knew what might be waiting for me on the other side of a door. Early in August, Chuck drove me to a private home in South Miami. The door was answered by a fat man with oiled black hair, maybe fifty-five years old. The fat man—his name was Leonard Campagno, also known as Lenny Camp—lived in incredible squalor.

His living room was filled with boxes and crates.

Newspapers were a carpet over the floor and cats were everywhere. Dishes with food still on them were spread over the table and piled in the sink. I could see cat hairs in the sugar bowl. Lenny led us through this litter to a bedroom in the back. It was not quite as sleazy as the rest of the house. At least there were clean sheets on the bed. Floodlights had been set up around the bed and were pointing down at it. Sometimes I felt almost invisible.

Now move it. I should never let anyone take my picture, and I should never sign my name to anything. He said those were two things that would always come back and haunt you later on.

There never was a way to argue with Chuck, no way to even discuss anything. I had no notion of what was going to happen here, only that I was going to take my clothes off and be photographed. In all our time together, Chuck never bothered to explain anything. Mine was no longer one of those lives where you could tell what was going to happen next. I was in the bathroom, naked, putting on some lipstick, when the door opened behind me.

My visitor was a tiny girl, five feet tall and very slim, brown-haired, about eighteen years old and also naked. All the time she put on her makeup, she was telling her life story. She glanced at me in the mirror and stopped putting on her lipstick. I had already figured out that I was going to be photographed in compromising positions—but which compromising positions? That was my only answer. Chicklet came over to me and put her arm around my shoulder. Suddenly her voice was very soothing.

Lenny tells us what he wants and we just go through the motions. So relax, willya? The fat man, two different cameras roped around his neck, was waiting for us. Chuck was leaning against a wall, staring right through me. I think his whole pleasure in life was watching me react to things. Just a few little kisserinos. As that was happening, I made myself go numb. I thought of myself as a metal robot, no human feelings at all, and that worked for a while.

I was feeling nothing. A skinny naked girl was kissing me on the mouth and I felt nothing at all. Click, click, click. No, no, no! Wouldja at least try to make it look natural? Try to make it look natural? What on earth would make that natural? I could be a robot through the kissing and even through touching her breast. I could go through the motions and erase them from my mind almost as they were happening. But when it came to something like sixty-nine, something that personal with another woman, it really blew my mind.

I had never even let a man do that to me. Look, when you go down on me, just fake it. All I know is that it was far worse. At the same time I was wondering what they were doing with the pictures they were taking. Where would they go? Would my mother and father ever see them? I had an awful feeling that the pictures would someday be used against me. Whether they were or not, they made this part of my life real, part of some record, uneraseable. This was a day of many firsts. The first photographs.

The first sex with another woman. And there was still another first to come. When the fat photographer had enough pictures of the two of us together, he went into another room and came back with something that he started to strap onto Chicklet.

It was a strap-on, make-believe, male sex organ. My first dildo. Then, assuming the male role, something she did with no difficulty at all, Chicklet got on top of me and put the dildo inside of me. The fat man came in very close with his camera. So there I was, being photographed by a fat degenerate while a skinny little girl with a make-believe penis was having sex with me, and I looked over at Chuck.

He was watching the scene with a very superior expression.

At moments like these, the lowest spots in my life, whenever I saw Chuck watching me, I would become aware of just one thing, his missing finger. Under normal circumstances—say we were just driving along in his car—I would never notice it. Now all I could see was the deformed hand, the absent finger.

Chicklet was my first female sex partner. Not my last. As soon as Chuck saw how much pain this experience caused me, he made sure that it became a key part of my repertoire. Many men like to rent that particular fantasy—two women making love to each other while the man becomes excited and then joins in. I only liked one of the women I was coupled with—and the sex itself had nothing whatever to do with my liking her.

Melody was the first person to help me in any way. One day she saw Chuck hit me and that was enough for her. From that moment on, she was my friend. Melody was a strange kind of a girl to be a hooker. She was very intelligent and always carried a book to read with her, even on jobs.

Melody was cute rather than sexy looking—about five-two, light brown curly hair, pug nose, non-stop smile. She reminded me of a high school cheerleader. Melody was a madame with a small operation of her own; she lived in and worked out of a tastefully decorated four-room apartment. If they were really weird, disgustingly so, Chuck made sure that they eventually found their way to me.

From the beginning, Melody seemed to take a personal interest in me. Whenever she had a job that required more than one hooker, she would call up Chuck and put in a request for me. Chuck would drive me to the meeting and wait nearby. He was never far away, but at least I was out of his sight for a time. Melody seemed to understand Chuck very well. She knew just what to tell him to get me off on a job with her.

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She would tell Chuck the most degenerate stuff she could think up and he would leap at the bait. Well, Linda would like to do that trick. This was an old guy in a wheelchair, barely able to stand up. He would hire six or seven girls and each girl would get a fifty-dollar bill. One girl would stand behind him, holding him upright, while all the other girls except me would be on the carpet, kissing each other and making love. My job was to kneel in front of him and suck him off.

Melody would give me that task because she knew that I found that easier than being with another woman. But it was no use; there was no holding them back. I told Melody everything that had happened: Tell him that today I tied all the other girls together, locking them into strange positions, and then the old guy began beating on everyone with a cane. Hit me! Harder, harder! Melody told me every unnatural act she had ever heard of, and then she made up some brand new ones just for Chuck.

And almost every day she managed to find me a trick in a hotel or an apartment, a job that got me away from Chuck, if only for a little while. Chuck was happy to have me do that one for two reasons: To make up for jobs like that one, Melody would also take me along on her easiest tricks.

There was Leo, a retired clothing manufacturer who had a condominium on the beach. When you first arrived, Leo made you go through closets filled with dresses, gowns, and lingerie. You were allowed to select whatever you liked. Afterwards, he would give us each thirty dollars and the clothes we had modeled; that clothing was the only payment I was ever allowed to keep.

I knew that someday God would get me away from Chuck but until then, until God made His move, my only help came from Melody. She kept thinking up new perversions for me to tell Chuck. She was like that girl in The Arabian Nights, the one who had to tell the king a brand new story every day, only the stories Melody told me were not exactly fairy tales. The two-headed dildoes are about this long and this big around.

Tell him that the trick had one of those with him and you had to insert both ends of it at once. I was sure that Chuck would start laughing at me when I told him a story like that. Every time Melody and I turned a trick together, we would find time for a little talk.

The first time Chuck saw me looking at it, he tore it from my hands and threw it away. There were other times with Melody when I felt very uncomfortable. One day after our regular session with Leo, she reached out and touched my hand. I do love you. Do you know what I mean?

I need you as a friend. You would learn to love me. She cared and that was enough. Maybe she was doing things for her own benefit. But whatever her motives were, she did manage to get Chuck Traynor off my back and out of my sight from time to time. Escape seemed a possibility. And escape was all I was thinking about those days. They wonder whether it is possible to go through weeks and months of incredible sexual activity and receive no sensual pleasure at all.

Did I enjoy any of it? I want to state this as clearly as I can. There was no pleasure. There was no love, no affection, no normal sex with anyone from the day I met Chuck Traynor until the day I finally got away. I did not have a single orgasm for six or seven years. I never had any enjoyment from any of it at all.

In fact, the only trick I could ever have a decent conversation with was a mortician named Jason. The first time Jason came to the house he told he just wanted to talk to me, nothing more. But for today I just wanted to get to know you. That was just fine with me. Even though he was a paying customer, Jason never did a cruel thing to me. At first, the fact that he was a mortician gave me the willies but that feeling faded as I spent more and more time with him.

No, it was never a pleasure—but it was a relief. One day, as his hour of romance was coming to an end, Jason made the mistake of asking me a question I had already heard a dozen times. I could tell that this was not the kind of thing he was paying to hear. As the story poured out of me, his mood went from serious to sad to deeply concerned. What woods? We could be together all the time and really be in love.

Perhaps I was getting into something far worse? What kind of man prefers make- believe love over real love? Then I thought about his being a mortician—maybe he was one of those guys who liked dead bodies; Chuck had told me about them. Maybe he was just another super-freak who wanted to get me up in the woods of Georgia so that he could kill me. The bottom line: I chickened out.

When I do think about it how, when I go back to moments like that, I start to jam—my head gets all jammed up. Or with Melody? How could it possibly have been any worse than what happened to me? Was I so terrified that everything in life scared me? Life with Chuck never improved. I learned to settle for the smallest imaginable triumphs, the absence of pain or the momentary lessening of terror. In time, I learned to satisfy men like Chuck—men who got their kicks from pain.

I learned how to do this without suffering too much pain myself. I set about teaching all of my muscles to relax. It got so that I could relax any set of muscles at will. So when Chuck started putting his fist inside of me, I was able to relax and cut back on the pain. On the contrary, I would scream for mercy, and he would become hard and ejaculate almost instantly; then he would leave me alone for a while.

I was becoming quite a little actress. I learned that it was never enough to fake pain, you had to fake pleasure at the same time: In a strange way, even the sword-swallowing, deep-throat techniques that Chuck had taught me could work to my advantage.

There were times when Chuck would make me work parties with maybe fifteen men and two chicks. I was a virgin until I was almost twenty years old, and only a couple of men before Chuck had ever made love to me.

What I found most degrading was when a man put his thing inside of me and came. The thought of fifteen men doing that in one night was more than I could tolerate. I had a choice of which was better for me to do, which made me feel more comfortable. And sucking cock made me feel more comfortable than being fucked. Because of my ability to totally relax my throat muscles, I became very popular with men who were into oral sex.

Chuck was very pleased with this. He called it word-of-mouth advertising. I can see it in the face asking the question. The questioner always has the sure knowledge that this could never have happened to him or to her.

They would have been strong enough and smart enough and resourceful enough to have gotten away. In fact, if the truth be known, they would never have allowed themselves to get into this kind of predicament in the first place. Once, during a grand jury hearing in California, I was asked the question point-blank: Even when no gun could be seen, there was a gun pointed at my head. I can understand why some people have such trouble accepting this as the truth. When I was younger, when I heard about a woman being raped, my secret feeling was that that could never happen to me.

I would never permit it to happen. But I know that it did happen once, and I know something else: It could happen again—to me or to you.

At first I was certain that God would help me escape but in time my faith was shaken. I became more and more frightened, scared of everything. The very thought of trying to escape was terrifying. I had been degraded every possible way, stripped of all dignity, reduced to an animal and then to a vegetable.

Whatever strength I had began to disappear. Simple survival took everything; making it all the way to tomorrow was a victory. The experience has enabled me to understand many events that others seem to find incomprehensible.

I have no difficulty relating to what happened to Patty Hearst; I have the feeling that we could be the closet of friends. Recently when several Playboy bunnies in Great Gorge, New Jersey, were drugged, photographed and forced to work as whores, I could understand the process. I can even comprehend the Jonestown massacre, hundreds of people standing in line, waiting to drink their cyanide.

I know what inhuman doses of fear and pain can do to any human being. Still, there were several times when I did try to escape. My first opportunity came after I had been working as a hooker for almost a month. Kitty— blonde, thin, intense and streetwise—had worked for Chuck earlier and was now returning after a year out on the streets, returning and bringing her own string of steady customers with her.

Kitty was tough and independent. When she realized that I was working against my will, she seemed sympathetic. Whenever his sister went away for a visit, Albert would get on the phone to Kitty.

Whenever I went to a new apartment building or hotel, I mentally charted the possible escape routes. This was a two-story, U-shaped building with only a single entranceway.

You walked through a set of arches, then about fifteen feet into a lobby, then up one flight of stairs to the apartment. As Chuck parked the car in front of the building, I realized that I would be out of his range of vision for several moments both coming and going. Sudden panic.

I needed time, time to figure out a plan, but there was no time. There was only now. Kitty and I walked from the car toward the arches framing the entrance. By this time we were up at the top of the stairs and Albert was waving us into his apartment. He was a short man, thin with a protruding pot belly. His bald head was rimmed with black hair.

Albert was wearing an undershirt and a pair of shapeless old-man trousers. His apartment reminded me of the set of an old movie. The lamps were covered with heavy fabric shades, and the overstuffed sofa had white doilies on the arm rests.

The stink of cologne was everywhere: As we walked through the living room toward the bedroom, my eyes were darting everywhere looking for an exit. We followed Albert into his bedroom and he turned his back on us and lifted his undershirt over his head. We just have to make nice to my sweetie here. Is there a back way out of here? She knew as well as I did what Chuck would do if we came down without the money.

I managed to dial 0 for Operator and then his hand came crushing down on the receiver. You girls go and get out of here now and just leave bygones be bygones. I have never been so scared. Then I saw that Chuck had moved his car and was parked about a hundred yards away from the entranceway. He seemed to be dozing behind the wheel. There was a chance and I took it.

I felt that I had nothing to lose, there would be a beating no matter what. Chuck, hurry up! If Kitty had remained quiet just one minute more—even a half-minute more—I would have had a shot.

But before I reached the corner, Chuck Traynor caught up with me and his grip burnt itself into my upper arm. Once we were back in the car, Chuck asked me no questions. What I had done was self-explanatory. Besides, Kitty was only too anxious to fill in the details. She told Chuck what I had said on the way into the building, what I had told the old man, even how I had tried to call the police.

At first I gave Kitty the benefit of the doubt. She took too much relish in telling the full story. She was just making brownie points. She wanted to be numero uno with Chuck. Later, when Melody heard this story, Kitty got a comeuppance of sorts. She was blacklisted by all the other hookers in the Miami area. My punishment was somewhat harsher. Chuck dropped Kitty off and then he took me home.

I remember being icy with fear. However, whatever Chuck did to me that afternoon—the details—are gone from my memory. It was a day before I could walk again. And once I could walk, there was nowhere to go. What had been prison was now solitary confinement. It was that kind of a marriage. Chuck Traynor spent much of that summer preparing for his upcoming trial on drug-smuggling charges.

It seems that pounds of marijuana—bales of pot wrapped around coke, hash, speed, LSD and assorted pills—had been dropped in a wooded area south of Miami, not far from the town of Homestead. Chuck and a friend had been caught carrying their bales to their cars, and a third confederate—this was Worth Devore—had gotten away uncaptured.

Mandina seemed as slick as Chuck was crude—always immaculately dressed, flashy and glib. Mandina and Chuck had once been partners in a tiny airline that made daily runs to the Bahamas. Despite their many surface differences, the two men had much in common, as I was to learn. I disliked Phil Mandina at first sight. And, as time went on, this dislike was to ripen into hate.

We all knew, of course, that Chuck was guilty. I was just another piece of furniture to them, part of the couch in the background, ignored, never part of the conversation, but always listening.

Chuck was working out his alibi. And we were out there checking the fields to find a place for the sky-diving club to jump, a target area, you dig? A sky-diving club. Hmmm, not bad at all.

However, a sky-diving club would have to have members.

Do you think you can swing that? Let me speak to a few guys. What were you doing out there in those fields? Did you know that was marijuana? When he was told that full blame for the operation was going to be placed on his shoulders, Worth had a few bad moments. At this point, Mandina seemed to notice my presence for the first time.

Although few of them were willing to risk a perjury charge, one friend, a fireman named Bob Phillips, went along with the story. This same Bob Phillips was later given a bit part in Deep Throat. I should have known better. Chuck never paid for anything himself. As a result of my automobile accident, I still had a case pending in New York and the lawyer who had been representing me was already receiving settlement offers. As payment for his case, Chuck gave Mandina the right to handle my case.

I never received any of the settlement money myself. In their last meetings before the trial, Mandina and Chuck went over all the details. Worth Devore had been instructed not to go near the courthouse under any circumstances.

Bob Phillips was ready to testify about the sky-diving club. And then Mandina put Chuck through his dress rehearsal, firing one question after another at him. It was not until the next day that I received my first serious proposal of marriage.

Marriage has always been important to me, perhaps too important. From when I was a small girl I had imagined what marriage would be like. That was all I ever expected from life—to get married to a good man, to have children, and to someday have a home of my own. When I got married, that was going to be it. Marriage is so important to me that I used to fantasize about all aspects of it—the proposal, the wedding night, the honeymoon. I had even imagined a man on his knees, asking for my hand.

Then the kicks started. The following morning we were married. There was no reason to remember the date; I knew that no anniversary would ever be celebrated. Chuck woke me up that day before dawn.

It was a six-hour drive to the small town of Valdosta, Georgia, a long drive made longer still by being locked in a car with Chuck.

Chuck was no longer content to get into a car and just drive somewhere. Now he had invented little games that helped him pass the time. One of his favorite games was to make me bare my breasts so that he could watch the reaction of passing truck drivers. And there was another game.

Teresa Silva

Before stopping for gas, he would hike my skirt up above my hips and make me spread my legs. He went to a novelty shop. The ring he selected was plastic, one of those interlocking puzzle rings made from a dozen strangely shaped pieces.

It set him back two dollars and change. For some reason, the ring did not seem at all inappropriate to the occasion. When we arrived in Valdosta, we went to the town hall. We were ushered into the office of a Justice of the Peace. The office had high ceilings, narrow windows, a wall of legal books. The ceremony itself was the simple economy model: The justice spent several minutes trying to get the pieces back together but had to give up.

Two aqueous solutions of PA nanofibres labelled with either Cy3 or Cy5 were mixed together and immediately adsorbed onto a glass coverslip at room temperature, freezing all kinetics. Therefore, single fibres should be either fully green or fully red. On the contrary, as shown in Fig. It is also possible to observe that some of the fibres visible in the green channel are not perfectly aligned with the overlapping fibres visible in the red channel Fig.

This provides further evidence that at this concentration bundles are observed instead of fully mixed fibres. Therefore, STORM allows us to verify the absence of fibre bundling and therefore select the optimal sample preparation to perform the molecular exchange experiments. Figure 4: Single fibre attachment.

Attachment was performed immediately on a glass coverslip after dilution NaCl 1 M. Full size image Molecular exchange kinetics and mechanisms The ability of STORM to image individual nanofibres with high resolution and to study the distribution of different molecular species inside aggregates vide supra , makes it the perfect tool to study exchange in PA samples.

We prepared samples for these experiments in similar manner to the FRET measurements. Aliquots of the solution were withdrawn over the course of 48 h and nanofibres were adsorbed onto a glass coverslip at room temperature to freeze the exchange. The better reconstruction of the fibres in the green channel reflects the selection of initially Cy3-labelled fibres for this experiment.

Full size image We verified that the exchange process is bidirectional by following green fibres incorporating red monomers Supplementary Fig. The intermediate time points provide insight into the exchange mechanism. This finding suggests an exchange mechanism on the basis of expulsion and reinclusion of PA molecules.

Polymerization—depolymerization at the nanofibre ends would have resulted in preferential exchange in those regions, while fragmentation—recombination would have resulted in a block-like structure; neither of these mechanisms is consistent with the acquired STORM images. To confirm this observation and further quantify the exchange, we studied the change in distribution of both dyes with time in initially green-labelled nanofibres, thus tracking the insertion of Cy5-PA.

Figure 6 shows the auto- and cross-correlation plots for the green and red channels at the different time points. The green channel displays the previously observed clustered distribution described by equation 2 rendering it less amenable for quantitative analysis.

Therefore, we monitored the autocorrelation of the red channel in time to analyse monomer exchange. The red channel autocorrelation, shown in Fig.

However, for later time points that is, after incubation longer than 6 h the quality of the fit deteriorates, indicating some non-random variations of the Cy5-PA concentration in the nanofibres other than the single decay originated from overcounting. Since clustering is not observed in single-colour Cy5-PA nanofibres Fig.

This heterogeneity can also be perceived visually by observing single fibre images Supplementary Fig. The cross-correlation between both channels was computed to assess how the distribution of one labelled PA would affect the distribution of the other, as shown in Fig. A cross-correlation of zero was found for the entire range, indicating that the distribution of Cy5-PA is not influenced by the presence of Cy3-PA, and vice versa.

In other words, the heterogeneous exchange of Cy5-PA is not due to the pre-existing clusters of Cy3-PA, but rather a consequence of structural variation in the PA nanofibre. Figure 6: Correlation of cyanine dyes along PA nanofibres initially only labelled with Cy3 during molecular exchange. Averaged autocorrelation plots for a Cy3 and b Cy5-labelled PAs and c averaged cross-correlation between both dyes. Full size image The observation of this heterogeneity in exchange, not detected by the FRET experiment, was possible due to the ability of STORM to evaluate the progress of molecular exchange for each individual supramolecular nanofibre.

The variability of the exchange progress between different aggregates could be measured, thus allowing us to further probe the heterogeneity in the system during and after molecular exchange. The linear density of localizations, defined as the number of localizations per nanometre arc length, has been computed and could be used as a rough estimate for the ensemble concentration of labelled molecules The density of Cy5-PA in originally single-colour green fibres was measured as a function of time, showing an increase in the average value as molecular exchange proceeded Fig.

However, the standard deviation also increased markedly, another indication for the presence of intrinsic structural diversity. In Fig. It was possible to observe a large subset of fibres that had a Cy5-PA density consistent with full mixing Fig. The existence of regions displaying minimal or no exchange after 48 h implies that full equilibration of these regions will take weeks to months, making them persistent for most practical purposes.

This analysis suggests that the population of fibres is considerably more heterogeneous after 48 h than would be expected on the basis of the FRET ensemble measurements. The equilibrium guideline is estimated from the localization density of original single-colour red fibres. Full size image Previous molecular dynamics simulations of a similar PA provide a theoretical framework for this structural diversity In these simulations, a broad distribution of secondary structures was found in the equilibrated fibre.

The heterogeneous molecular exchange pattern observed in our study might stem from this conformational diversity. On the other hand, the surface moieties in these nanofibres have been identified by the electron paramagnetic resonance experiments as regions where liquid-like dynamics prevail.

This is in contrast to supramolecular systems with weaker internal cohesion that could exchange molecules to produce completely homogeneous environments The formulation of supramolecular systems with high levels of internal order and cohesion such as the PA nanofibres will therefore open new avenues to generate structural diversity for functional purposes. One example will be their ability to adapt structurally to bind important bioactive targets.

Remarkably, the coexistence of fully dynamic and kinetically inactive areas in the aggregate architecture was observed, demonstrating the existence of structural diversity in PA nanofibres. This intriguing dynamic behaviour is foreseen to have important implications in the biological performance of supramolecular systems. Methods Peptide synthesis and purification PAs were synthesized using standard Fmoc solid-phase peptide synthesis.

Cyanine dyes were supplied by Cyandye USA. ACS-grade solvents were used for synthesis. For each amino acid coupling, 4. The coupling cocktail was then added to 0. Finally, the palmitic acid tail 8. Cleavage solution was concentrated by rotary evaporation and the PA precipitated in cold diethyl ether.

All eluents and additives were HPLC grade. The rest of the synthetic procedure proceeded as described above. Cy3- and Cy5-labelled PAs were cleaved and purified as described above. Lyophilization yielded a red powder for Cy3 and blue for Cy5. Peptides, cyanine dye-coupled peptides and eventual contaminants were detected using a PDA Shimadzu SPD-M20A , acquiring a full ultraviolet-visible spectrum between and nm at any time point.

For that, samples were manually injected bypassing the column at a flow rate of 0. Similar to other anionic PA systems, these formed hydrogels in the presence of CaCl2. The degree of labelling of the fibres was accurately controlled by simply mixing PA stock solutions at the desired ratio.

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Solutions of non-labelled PA were mixed with aqueous solutions of either one or both labels at certain mole ratios, flash-frozen in liquid nitrogen and lyophilized. The obtained material was redissolved in NH4OH aq. These two last steps resembled the cleavage and purification conditions, respectively.

These steps were undertaken to provide molecular mixing, as well as to reset PA self-assembly history. Non-labelled PA was treated using the same method before measurements where the fluorescence-labelled molecules were absent. The obtained powders were reconstituted in appropriate buffers before measurements.

After the harsh acidic conditions dissolution in TFA used to create a homogenous molecular mixture, PAs were checked by analytical HPLC to rule out potential degradation of the original molecules. Analytical HPLC was performed as described above. Supplementary Fig. The FRET intensity increased with concentration of acceptor. This indicates that the sample preparation is suitable to assure co-assembly of the three PAs in a single supramolecular object. Circular dichroism spectroscopy Circular dichroism spectra were recorded using Jasco Circular Dichroism Spectrometer model J PA was dissolved in water at 10 mM.

Before each measurement, PA stock solution was diluted in appropriate buffers to yield a final PA concentration of 0. A quartz cuvette of 1 mm path length was used for the measurements. Each trace represents the average of five scans. For imaging, the sample was transferred to a Gatan cryo-holder under liquid N2, and images were obtained with a Gatan CCD camera. Critical micelle concentration by Nile Red assay Nile Red is a hydrophobic solvatochromic dye water soluble at low concentration with high affinity to the hydrophobic pocket of micelles.

In aqueous environment, the molecule is weakly fluorescent. In hydrophobic environments, fluorescence intensity is around times higher and a pronounced blue shift is observed. Therefore, Nile Red is commonly used to probe the existence of hydrophobic pockets formed by surfactants in aqueous environment PA solution was diluted serially to obtain concentrations in the range nM mM.

Solutions were aged for 24 h to assure full nanofibre disassembly at concentrations below the critical micelle concentration. Fluorescence-emission spectra excitation nm were recorded for an emission range between and nm. The maximum intensity and respective wavelength at maximum intensity were both represented as a function of the logarithm of the PA concentration.

At concentrations close to critical micelle concentration, it should be observed a sharp increase in fluorescence intensity and a hypsochromic effect. Emission spectra were acquired using excitation wavelengths of or nm for Cy3 or Cy5, respectively. Excitation spectra were collected using emission wavelengths of or nm for Cy3 or Cy5, respectively. Fluorescence-emission spectra of dual-labelled PAs were recorded at the excitation wavelength of Cy3 nm, at which direct excitation of Cy5 is negligible.